Method for biosynthesizing acetyl tetrapeptide-5, composition containing acetyl tetrapeptide and application of composition

Abstract

The invention relates to the technical field of polypeptide biosynthesis, in particular to a method for biosynthesizing acetyl tetrapeptide-5, a composition containing the acetyl tetrapeptide-5 and application of the composition. According to the method, low-cost natural amino acid is used as a substrate, specific aminopeptidase or a mutant thereof is used as a biocatalyst, amino acid ligase and polyphosphatase are combined, and acetyl tetrapeptide-5 can be efficiently synthesized through a one-step enzymatic reaction. Compared with a traditional chemical synthesis method, the method has the advantages of low raw material cost, no use of an organic solvent, mild reaction conditions, high regioselectivity, environmental friendliness, low production cost and the like. The invention also provides a composition containing acetyl tetrapeptide-5 and dipeptide-15, the inhibition effect of the composition on grease secretion is obviously better than that of a single component, and the composition has a synergistic oil control effect under a specific ratio.

Description

Technical Field

[0001] This invention relates to the field of polypeptide biosynthesis technology, and more particularly to a method for biosynthesizing acetyl tetrapeptide-5, compositions containing acetyl tetrapeptide-5, and their applications. Background Technology

[0002] Sebum is primarily produced by sebaceous glands, and its synthesis is regulated by multiple factors, including endocrine factors (androgens, etc.), metabolic enzymes (such as fatty acid synthase FASN), local inflammation, neuroendocrine signaling, and the skin microbiome. Current oil-control strategies typically target: physical adsorption of excess sebum, inhibition of biochemical pathways in sebum synthesis, regulation of keratin / pore condition, or inhibition of sebum-related inflammation / microbial activity. 1 .

[0003] Excessive sebum secretion can lead to skin barrier dysfunction, clogged pores, and an oily feel, increasing the risk of skin inflammation and acne formation. It also promotes the overgrowth of microorganisms (such as Propionibacterium acnes), exacerbating acne, pimples, and skin sensitivity. Long-term excess oil can also cause thickening of the stratum corneum, resulting in rough skin, uneven skin tone, and makeup that easily comes off. Furthermore, it accelerates oxidative stress in the skin, inducing inflammation and premature aging, affecting overall skin health and appearance.

[0004] Chinese patent CN119656057A discloses a novel application of acetyl tetrapeptide-5 in the preparation of oil-controlling cosmetics. Cellular experiments have verified that it can significantly reduce the lipid content of human sebaceous gland cells (SZ95), with a maximum inhibition rate of 87%, demonstrating a significant oil-controlling effect.

[0005] Patent CN101061135A discloses a method for solid-phase synthesis of acetyl tetrapeptide-5. This method involves condensing Fmoc-L-His(Trt)-OH into a 2-chlorotriphenylmethyl resin, followed by sequential deprotection and coupling of Fmoc-L-Ser(tBu)-OH, Fmoc-L-His(Trt)-OH, and Fmoc-βAla-OH to obtain Fmoc-βAla-L-His(Trt)-L-Ser(tBu)-L-His(Trt)-resin. Further deprotection and cleavage yield acetyl tetrapeptide-5 (βAla-His-Ser-His). However, this method uses expensive raw materials such as the 2-chlorotriphenylmethyl resin and the side-chain protected amino acids. Furthermore, the condensation and coupling must be carried out in large quantities of organic solvents such as DMF, increasing production costs and potentially causing environmental pollution. The subsequent cleavage and purification processes also consume large amounts of reagents, resulting in a cumbersome process with low yield, making it difficult to meet the needs of large-scale industrial production. Therefore, it is of great significance to develop an efficient, green, and low-cost biosynthetic method to prepare acetyl tetrapeptide and promote its widespread application in cosmetics, pharmaceuticals and other fields.

[0006] US Patent 20230320967A discloses a novel application of a proline-containing dipeptide compound—dipeptide-diaminobutyryl benzylamine diacetate—in regulating sebaceous gland function and controlling oil production. As a DPP4 inhibitor, it can inhibit sebaceous cell proliferation, reduce neutral lipid production, and significantly improve oiliness, enlarged pores, and acne problems. Clinical studies show that continuous use for 28 days can effectively reduce sebum secretion and skin radiance, demonstrating significant oil control and skin-beautifying effects.

[0007] Chinese patent CN116744896A discloses a skincare composition containing palmitoyl dipeptide-7 (Pal-KT) and acetyl tetrapeptide-11 (Ac-PPYL). These two ingredients synergistically upregulate PPARA and MSMO1 gene expression in a ratio of 10:1 to 1:10, increasing cellular ATP levels, promoting cellular energy metabolism and ECM repair, and improving skin aging problems such as wrinkles, sagging, and barrier damage. The composition is highly effective even at low doses.

[0008] Chinese patent CN1886114B discloses a series of specific amino acid derivatives (including dipeptide-15) and their salts, which significantly inhibit parakeratosis, shrink pores and improve skin roughness, thereby forming parakeratosis inhibitors, pore shrinking agents and their topical compositions with such compounds as the main active ingredients, which are widely used in facial and body skin care preparations.

[0009] While the above compositions have shown potential and advantages in oil control and anti-aging, they still have limitations: Acetyl tetrapeptide-5 has a single target, controlling oil production only by inhibiting lipid production, with limited effects on sebum-related inflammation or microbial regulation; proline dipeptide derivatives have a long clinical use period, with no obvious short-term effects, and only target DPP4, lacking multi-pathway synergistic effects; the combination of palmitoyl dipeptide-7 and acetyl tetrapeptide-11 mainly improves aging and the skin barrier, with insufficient oil control; dipeptide-15 and its derivatives are biased towards keratin regulation, with limited inhibition of sebum production, and have a single function; combinations of multiple plant extracts have complex components, poor stability, unclear mechanisms of action, and potential irritation risks. Overall, existing technologies still have shortcomings in terms of oil control efficiency, breadth of action, safety, and scientific basis, making it difficult to achieve a gentle, non-irritating, highly effective, and synergistic oil control effect. Summary of the Invention

[0010] In view of this, the present invention provides a method for biosynthesizing acetyl tetrapeptide-5, a composition containing acetyl tetrapeptide-5, and their applications. The present invention uses natural amino acids as substrates and can efficiently synthesize acetyl tetrapeptide-5 through a one-step enzymatic reaction, resulting in low synthesis cost and high yield. Furthermore, the composition comprising acetyl tetrapeptide-5 and dipeptide-15 provided by the present invention exhibits a synergistic oil-controlling effect under specific ratios.

[0011] The method for synthesizing acetyl tetrapeptide-5 includes the following steps:

[0012] S1. Under the catalysis of aminopeptidase or its mutant, N-acetyl-β-alanine methyl ester or its salt is reacted with L-histidine to obtain filamentous dipeptide and acetylcarnosine.

[0013] S2. Add adenosine monophosphate, inorganic polyphosphate, polyphosphokinase and amino ligase to the reaction system of step S1, and generate acetyl tetrapeptide-5 through reaction.

[0014] In this invention, the aminopeptidase is derived from at least one of the following: *Ajellomyces capsulatus*, *Colletotrichum gloeosporioides*, *Eutypa lata*, *Beauveria bassiana*, *Cyphellophora europaea*, *Pyricularia oryzae*, and *Gaeumannomyces tritici*.

[0015] In some preferred embodiments, the aminopeptidase comprises at least one of the following:

[0016] (1) The enzyme Car02, derived from Ajellomyces capsulatus with UniProt ID accession number C6H1Y6;

[0017] (2) Enzyme Car03 derived from Colletotrichum gloeosporioides with UniProt ID accession number T0M6U4;

[0018] (3) Enzyme Car05, derived from Eutypa lata with UniProt ID accession number M7SYE6;

[0019] (4) Enzyme Car06, derived from Beauveria bassiana with UniProt accession number J4UIY7;

[0020] (5) Enzyme Car08 derived from Cyphellophora europaea with UniProt ID accession number W2S4G1;

[0021] (6) Car09, an enzyme derived from Pyricularia oryzae with UniProt accession number G4NHJ4;

[0022] (7) Car011, an enzyme derived from Gaeumannomyces tritici with UniProt ID accession number J3PFY1.

[0023] This invention investigated the catalytic effects of aminopeptidases from different sources. The results showed that all seven sources exhibited good catalytic activity towards their substrates. Among them, *Colletotrichum gloeosporioides*, *Eutypa lata*, *Beauveria bassiana*, and *Pyricularia oryzae* demonstrated higher catalytic activity and substrate specificity in catalyzing the reaction of N-acetyl-β-alanine methyl ester with L-histidine to generate filamentous dipeptides and acetylcarnosine, significantly improving the conversion rate of the first step reaction and the yield of the target product.

[0024] In the reaction catalyzing the reaction of N-acetyl-β-alanine methyl ester with L-histidine to generate filamentous dipeptide and acetylcarnosine, an aminopeptidase mutant can also be used for catalysis. The aminopeptidase mutant has an amino acid sequence as shown in any of the following: (a) an amino acid sequence as shown in SEQ ID NO: 32; (b) an amino acid sequence obtained by substitution, deletion, addition, or modification of one or more amino acids in the sequence shown in (a); (c) an amino acid sequence having at least 80% identity with (a) or (b); (d) an aminopeptidase having at least 80% sequence identity with at least one of (a) to (c) aminopeptidases.

[0025] This invention involves mutating the wild-type aminopeptidase Car06 (amino acid sequence shown in SEQ ID NO: 32). The resulting mutants Car06_C85S, Car06_C85A, Car06_T184V, Car06_K262A, Car06_N305G, Car06_N305V, and Car06_N305F all showed significantly improved efficiency in catalyzing the production of carnosine compared to the wild type. Among them, the mutant Car06_N305F, for example, exhibited the highest catalytic activity.

[0026] For the solvent used in the reaction described in step S1, the present invention preferably includes at least one of water, phosphate buffer, Tris-HCl buffer, and HEPES buffer, wherein the concentration of the buffer is preferably 20-100 mM, and the pH range is preferably 7.0-9.0. These buffers can provide a more stable pH environment for the enzymatic reaction, ensuring the catalytic activity of aminopeptidase or its mutant.

[0027] In some embodiments, step S1 further includes the following steps for preparing the aminopeptidase or its mutant: cloning a nucleic acid molecule encoding the aminopeptidase or its mutant into a vector, transforming it into a prokaryotic expression system, culturing and inducing expression in the prokaryotic expression system, and then separating and purifying to obtain a crude enzyme solution containing the aminopeptidase or its mutant. The sequence of the nucleic acid molecule is codon-optimized according to the host type of the prokaryotic expression system. The host includes *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, *Pichia pastoris*, or *Streptomyces*, preferably *Escherichia coli*. In a specific embodiment of the present invention, the host is *Escherichia coli* BL21(DE3). The backbone of the recombinant vector includes pET series, pQE series, pHT series, pXMJ19, pHIL series, pPIC series, or pSET152 vectors. *Escherichia coli* commonly uses pET and pQE series vectors; *Bacillus subtilis* commonly uses pHT series vectors; *Corynebacterium glutamicum* commonly uses pXMJ19 vectors; *Pichia pastoris* commonly uses pHIL and pPIC series vectors; and *Streptomyces* commonly uses pSET152 vectors. In a specific embodiment of the present invention, the backbone vector is a PET series vector, specifically pET-28a or pET-30a. During the culture process, when the concentration of *E. coli* reaches a certain level, IPTG (isopropyl-β-D-thiogalactoside) is added to induce expression. The specific induction conditions can be optimized and adjusted according to different recombinant vectors and host bacteria. After the induction expression is completed, the bacterial cells are collected by centrifugation and disrupted by ultrasonication to obtain a crude enzyme solution. To improve the catalytic efficiency of aminopeptidase or its mutants, the crude enzyme solution can be further purified by ion exchange chromatography or other purification methods to obtain a high-purity aminopeptidase or its mutants.

[0028] In step S2, the adenosine monophosphate compound includes adenosine monophosphate (AMP), adenosine triphosphate disodium salt (ATP-Na2), or adenosine diphosphate disodium salt (ADP-Na2), wherein the concentration of the adenosine monophosphate compound is preferably 1-5 mM, specifically 1 mM, 2 mM, or 5 mM. The polyphosphate includes sodium hexametaphosphate and / or sodium tripolyphosphate. The molar amount of sodium hexametaphosphate is the molar amount of the substrate filament dipeptide or acetylcarnosine divided by 3-3.5. Further, the concentration of sodium hexametaphosphate is preferably 26-30 mM, specifically 26 mM, 28 mM, 30 mM, or any value within the above range. The amino acid ligase is derived from *Ralstonia solanacearum*; in a specific embodiment of the invention, the amino acid ligase is Rsp1486 from *Ralstonia solanacearum*. The polyphosphoric acid kinase is derived from *Cytophaga hutchinsonii*; in a specific embodiment of the invention, the polyphosphoric acid kinase is ChPPK2 (∆285-305) from *Cytophaga hutchinsonii*. In step S2, the reaction conditions include a reaction temperature of 32-45℃ for 1-6 hours, specifically 32℃, 37℃, 40℃, or 45℃, and a reaction time of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. These reaction conditions are beneficial for the function of each enzyme and can further improve the yield of acetyl tetrapeptide-5. In addition, an activator of polyphosphokinase, such as magnesium ions, can be added in step S2, which can significantly improve the catalytic efficiency of polyphosphokinase, accelerate the conversion of polyphosphate to ATP, and thus promote the smooth progress of the second step reaction.

[0029] Furthermore, the present invention provides an aminopeptidase mutant that mutates aminopeptidase Car06 (amino acid sequence as shown in SEQ ID NO: 7), the mutant including at least one mutation site selected from C85S, C85A, T184V, K262A, N305G, N305V, and N305F. Further, the mutation site of the mutant is any one of C85S, C85A, T184V, K262A, N305G, N305V, and N305F, and the corresponding mutants are sequentially named Car06_C85S, Car06_C85A, Car06_T184V, Car06_K262A, Car06_N305G, Car06_N305V, and Car06_N305F. The mutant Car06_N305F has an amino acid sequence as shown in any of the following:

[0030] (a) The amino acid sequence as shown in SEQ ID NO: 32;

[0031] (b) An amino acid sequence obtained by substitution, deletion, addition or modification of one or more amino acids in the sequence shown in (a);

[0032] (c) An amino acid sequence that is at least 80% identical to (a) or (b).

[0033] The present invention also provides biomaterials, characterized in that they comprise any one of the following:

[0034] 1) Nucleic acids having sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, i.e., nucleic acids encoding aminopeptidases Car02, Car03, Car05, Car06, Car08, Car09 or Car011;

[0035] 2) Nucleic acid encoding the mutant as described above;

[0036] 3) An expression cassette containing the nucleic acid described in 1) or 2);

[0037] 4) A recombinant vector comprising any one of the nucleic acids described in 1) to 2) or comprising the expression cassette described in 3);

[0038] 5) Transfect or transform the host of the recombinant vector described in 4).

[0039] The nucleic acid encoding the mutant refers to a DNA or RNA molecule capable of being transcribed and translated to produce the aforementioned aminopeptidase mutant. In some embodiments, the nucleic acid encoding the mutant is DNA, specifically a nucleic acid codon-optimized according to the host. Further, the optimized nucleic acid encoding the mutant includes the nucleic acid encoding any one of the mutants Car06_C85S, Car06_C85A, Car06_T184V, Car06_K262A, Car06_N305G, Car06_N305V, and Car06_N305F. Specifically, the nucleic acid is obtained by amplification using the nucleic acid shown in SEQ ID NO: 7 as a template and with any of the following primer pairs: SEQ ID NO: 14~15, SEQ ID NO: 16~17, SEQ ID NO: 18~19, SEQ ID NO: 20~21, SEQ ID NO: 24~25, SEQ ID NO: 26~27, and SEQ ID NO: 30~31. The nucleic acid encoding Car06_N305F (SEQ ID NO: 32) has the sequence shown in SEQ ID NO: 33, which is obtained by amplification using primers SEQ ID NO: 30-31. In this invention, the nucleic acid encoding the mutant also has: a nucleic acid sequence in which one or more bases have been substituted, deleted, added, or modified in the sequence of the nucleic acid described above; or a nucleic acid sequence having at least 80% identity with the sequence of the aforementioned nucleic acid. In this invention, all the above nucleic acid sequences are codon-optimized sequences based on *E. coli*. Codon optimization can also be performed according to different host expression systems (such as prokaryotic or eukaryotic hosts), and the optimized nucleic acid sequences can improve expression efficiency in specific hosts.

[0040] The biological materials provided by this invention include *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, *Pichia pastoris*, or *Streptomyces*, preferably *Escherichia coli*. In a specific embodiment of this invention, the host is *Escherichia coli* BL21(DE3). The backbone of the recombinant vector includes pET series, pQE series, pHT series, pXMJ19, pHIL series, pPIC series, or pSET152 vectors. *Escherichia coli* commonly uses pET and pQE series vectors; *Bacillus subtilis* commonly uses pHT series vectors; *Corynebacterium glutamicum* commonly uses pXMJ19 vectors; *Pichia pastoris* commonly uses pHIL and pPIC series vectors; and *Streptomyces* commonly uses pSET152 vectors. In a specific embodiment of this invention, the backbone vector is a PET series vector, specifically pET-28a or pET-30a vector.

[0041] The present invention also provides the use of the mutants or the biological materials described above in the biosynthesis of acetyl tetrapeptides.

[0042] The present invention also provides a composition comprising acetyl tetrapeptide-5 (T5) and dipeptide-15 (i.e., diglycinide, D15).

[0043] In this invention, the preferred mass ratio of acetyl tetrapeptide-5 to dipeptide-15 is 1:(150~200), specifically 1:150 or 1:200. Experiments show that the composition with this ratio significantly inhibits sebum secretion from sebaceous gland cells (SZ95) better than any single ingredient. Using the BILSS independent model to calculate the combined effect, the CI values ​​for the above ratios are all greater than 1, indicating that acetyl tetrapeptide-5 (T5) and dipeptide-15 (D15) have a significant synergistic effect at this ratio, achieving a synergistic oil-control effect. The CI value is highest and the synergistic effect is optimal when the mass ratio is 1:150. By synergistically regulating sebum production and keratin metabolism, the two components effectively inhibit shine, tighten pores, and improve rough skin texture, while also protecting the skin barrier and maintaining microecological balance. They are effective at low doses, have high safety, and meet the needs for rapid, comprehensive oil control and optimized skin health.

[0044] This invention provides a method for the biosynthesis of acetyl tetrapeptide-5, compositions containing acetyl tetrapeptide-5, and their applications. This invention uses low-cost natural amino acids as substrates, specific aminopeptidases or their mutants as biocatalysts, and combines amino acid ligases and polyphosphatases to efficiently synthesize acetyl tetrapeptide-5 through a one-step enzymatic reaction. Compared with traditional chemical synthesis methods, it has advantages such as low raw material costs, no use of organic solvents, mild reaction conditions, high regioselectivity, environmental friendliness, and low production costs. This invention also provides a composition comprising acetyl tetrapeptide-5 and dipeptide-15, which exhibits significantly better inhibitory effects on sebum secretion than the individual components, and demonstrates a synergistic sebum-controlling effect under specific formulations. Attached Figure Description

[0045] Figure 1 Electrophoresis image of aminopeptidase Car01-06 protein;

[0046] Figure 2 Electrophoresis image of aminopeptidase Car07-11 protein;

[0047] Figure 3 Electrophoresis image of the aminopeptidase Car06 autocleavage protein;

[0048] Figure 4 Electrophoresis images of amino acid ligase Rsp1486 and polyphosphoric acid kinase ChPPK2 (∆285-305);

[0049] Figure 5 This is the reaction formula for the enzymatic synthesis of acetyl tetrapeptide-5;

[0050] Figure 6 The effect of different ratios of T5 and D15 on inhibiting sebum secretion. Detailed Implementation

[0051] This invention provides a method for the biosynthesis of acetyl tetrapeptide-5, compositions containing acetyl tetrapeptide-5, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0053] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0054] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0055] In this article, when referring to units of data ranges, if the unit is only followed by the right endpoint, it means that the units of the left and right endpoints are the same. For example, 1~5mM means that the units of the left endpoint "1" and the right endpoint "5" are both in millimeter (mM).

[0056] The sequence information involved in this invention is as follows:

[0057] The amino acid sequence of Car01:

[0058] MERKRIRELLPNLRFGQFPTGPKNSLTDVPGVLVSTKSVIKPADLPHHHEVNTGVTTILPRKEWFKQGCYASYFRFNGSGEMTGSHWIDESGLLNSPVIITNSFGVGACYNGVYEYAKKHHKDEKGICDWFLTPVIAETFDGWLSDIGAMAVQSSDVVEGIENASSDAVPEGCTGGGTGMITMGFKAGTGNASRVIDSVKIDSKGEKQQVKYTLAALVQSNFGGARFLTVNGVPVGRILEDEAMAAKKAGPMDGPEGSIIVVIATDAPLIPIQLQRLAKRATVGVARTGGWGSNYSGDIFLAFSTAHEIPRENTQNWTPSVPQPQEVLDTESINALFEAAFEAVEEAIYNAICMATDTKGPDGREVKAIDLEKLKEIVTRHAY (SEQ ID NO: 1);

[0059] Nucleotide sequence of Car01 (codon-optimized):

[0060]

[0061] Nucleotide sequence of Car02 (codon optimized):

[0062]

[0063] Nucleotide sequence of Car03 (codon optimized):

[0064]

[0065] Nucleotide sequence of Car04 (codon optimized):

[0066]

[0067] Nucleotide sequence of Car05 (codon optimized):

[0068]

[0069] Amino acid sequence of Car06:

[0070] MTSTTSAQTNAKRVRLRELIPEIRLGKWERTGPKNGITDVKGVLVHTESIHDKEKGVNTGVTTIVPRKEWMEQACYAGMFRYNGCGELTGSHWLDDTGLLGSPIVLSNSFAVGACHTGIYQYGARHYRNQSGDIDWFLMPVVGETYDGMLNDLSQFVVTPEHVIRGIEKASSDPVPEGNTGGGTGMTCHGFKAGTGTSSRLVPGLDQDGKETDYTVAALVQANYGRLYSFTVAGAPVGRILWEEQGREAAAKKARDEYEKAKSIKDGSIIVVIATDAPLNPVQLQRLARRATRGVSRVGGYGNTNSGDIFMAFSTGTVAPGQYESGAIEQAPDRAELLQHKAVDERKMDSLLEAAGEATEEAILNAICMAETLTGFRGRTVEAMPLERVKNIVQRHIDLEKELAAKQN (SEQ ID NO: 7);

[0071] Nucleotide sequence of Car06 (codon-optimized):

[0072]

[0073] The nucleotide sequence of Car07 (codon optimized):

[0074]

[0075] The nucleotide sequence of Car08 (codon optimized):

[0076]

[0077] Nucleotide sequence of Car09 (codon optimized):

[0078]

[0079] The nucleotide sequence of Car10 (codon optimized):

[0080]

[0081] The nucleotide sequence of Car11 (codon optimized):

[0082]

[0083] Construct the nucleotide sequence of the upstream primer for the Car06_C85S mutant plasmid:

[0084] ATGTTTCGCTATAATGGTTCTGGCGAATTAACCGGTAGTC (SEQ ID NO: 14);

[0085] Construct the nucleotide sequence of the downstream primer for the Car06_C85S mutant plasmid:

[0086] ACTACCGGTTAATTCGCCAGAACCATTATAGCGAAACATC (SEQ ID NO: 15);

[0087] Construct the nucleotide sequence of the upstream primer for the Car06_C85A mutant plasmid:

[0088] GATGTTTCGCTATAATGGTGCTGGCGAATTAACCGGTAGTC (SEQ ID NO: 16);

[0089] Construct the nucleotide sequence of the downstream primer for the Car06_C85A mutant plasmid:

[0090] ACTACCGGTTAATTCGCCAGCACCATTATAGCGAAACATCC (SEQ ID NO: 17);

[0091] Construct the nucleotide sequence of the upstream primer for the Car06_T184V mutant plasmid:

[0092] GAATACTGGCGGCGGCGTCGGCATGACCTGTCATG (SEQ ID NO: 18);

[0093] Construct the nucleotide sequence of the downstream primer for the Car06_T184V mutant plasmid:

[0094] ATGACAGGTCATGCCGACGCCGCCGCCAGTATTCC (SEQ ID NO: 19);

[0095] Construct the nucleotide sequence of the upstream primer for the Car06_K262A mutant plasmid:

[0096] CGAATATGAGAAGGCCGCATCCATCAAAGATGGC (SEQ ID NO: 20);

[0097] Construct the nucleotide sequence of the downstream primer for the Car06_K262A mutant plasmid:

[0098] GCCATCTTTGATGGATGCGGCCTTCTCATATTCG (SEQ ID NO: 21);

[0099] Construct the nucleotide sequence of the upstream primer for the Car06_S268C mutant plasmid:

[0100] CAAATCCATCAAAGATGGCTGCATTATCGTCGTGATCGCGAC (SEQ ID NO: 22);

[0101] Construct the nucleotide sequence of the downstream primer for the Car06_S268C mutant plasmid:

[0102] TCGCGATCACGACGATAATGCAGCCATCTTTGATGGATTTGG (SEQ ID NO: 23);

[0103] Construct the nucleotide sequence of the upstream primer for the Car06_N305G mutant plasmid:

[0104] TTGGCGGCTACGGGAACACGGGTTCCGGTGATATTTTTATGGC (SEQ ID NO: 24);

[0105] Construct the nucleotide sequence of the downstream primer for the Car06_N305G mutant plasmid:

[0106] CCATAAAAATATCACCGGAACCCGTGTTCCCGTAGCCGCCAAC (SEQ ID NO: 25);

[0107] Construct the nucleotide sequence of the upstream primer for the Car06_N305V mutant plasmid:

[0108] TTGGCGGCTACGGGAACACGGTTTCCGGTGATATTTTTATGGC (SEQ ID NO: 26);

[0109] Construct the nucleotide sequence of the downstream primer for the Car06_N305V mutant plasmid:

[0110] CCATAAAAATATCACCGGAAACCGTGTTCCCGTAGCCGCCAAC (SEQ ID NO: 27);

[0111] Construct the nucleotide sequence of the upstream primer for the Car06_N305P mutant plasmid:

[0112] TTGGCGGCTACGGGAACACGCCTTCCGGTGATATTTTTATGGC (SEQ ID NO: 28);

[0113] Construct the nucleotide sequence of the downstream primer for the Car06_N305P mutant plasmid:

[0114] CCATAAAAATATCACCGGAAGGCGTGTTCCCGTAGCCGCCAAC (SEQ ID NO: 29);

[0115] Construct the nucleotide sequence of the upstream primer for the Car06_N305F mutant plasmid:

[0116] TTGGCGGCTACGGGAACACGTTTTCCGGTGATATTTTTATGGC (SEQ ID NO: 30);

[0117] Construct the nucleotide sequence of the downstream primer for the Car06_N305F mutant plasmid:

[0118] CCATAAAAATATCACCGGAAAACGTGTTCCCGTAGCCGCCAAC (SEQ ID NO: 31);

[0119] The amino acid sequence of the mutant Car06_N305F:

[0120] MTSTTSAQTNAKRVRLRELIPEIRLGKWERTGPKNGITDVKGVLVHTESIHDKEKGVNTGVTTIVPRKEWMEQACYAGMFRYNGCGELTGSHWLDDTGLLGSP IVLSNSFAVGACHTGIYQYGARHYRNQSGDIDWFLMPVVGETYDGMLNDLSQFVVTPEHVIRGIEKASSDPVPEGNTGGGTGMTCHGFKAGTGTSSRLVPGLD QDGKETDYTVAALVQANYGRLYSFTVAGAPVGRILWEEQGREAAAKKARDEYEKAKSIKDGSIIVVIATDAPLNPVQLQRLARRATRGVSRVGGYGNTFSGDIFMAFSTGTVAPGQYESGAIEQAPDRAELLQHKAVDERKMDSLLEAAGEATEEAILNAICMAETLTGFRGRTVEAMPLERVKNIVQRHIDLEKELAAKQN (SEQ ID NO: 32);

[0121] Nucleotide sequence of mutant Car06_N305F:

[0122]

[0123] The UniProt accession number for the amino acid ligase Rsp1486 derived from Ralstonia solanacearum is Q8XQ00. The codon-optimized nucleotide sequence is as follows:

[0124]

[0125] The UniProt accession number for the polyphosphoric acid kinase ChPPK2 derived from Cytophaga hutchinsonii is A0A6N4SMB5. The codon-optimized nucleotide sequence is as follows:

[0126] ATGGCCACCGACTTCTCGAAGCTGTCCAAATATGTAGAGACCCTGCGTGTTAAACCGAAACAGAGTATTGACTTGAAAAAAGATTTTGATACTGACTACGATCATAAAATGTTAACAAAAGAAGAAGGCGAGGAGCTGTTGAATCTGGGTATTTCCAAGCTGTCAGAGATTCAAGAGAAACTTTACGCGAGTGGAACGAAGTCTGTCCTGATCGTGTTTCAAGCGATGGATGCCGCGGGAAAGGACGGCACTGTTAAGCACATCATGACAGGGTTGAACCCACAGGGAGTGAAGGTGACCAGCTTTAAGGTACCTTCCAAGATTGAACTGAGCCACGATTATTTGTGGCGTCATTATGTAGCATTACCCGCAACGGGTGAGATCGGAATTTTTAATCGCTCCCATTACGAGAACGTGTTGGTGACTCGCGTCCACCCCGAATACCTGCTGAGCGAGCAAACCTCAGGGGTGACGGCAATTGAACAAGTTAATCAAAAATTCTGGGACAAGCGCTTTCAGCAGATCAATAATTTTGAGCAGCATATCAGTGAAAACGGAACGATTGTCTTGAAGTTCTTCCTGCATGTGTCAAAGAAGGAACAGAAGAAGCGCTTTATCGAACGCATTGAACTTGACACGAAAAATTGGAAATTCTCAACCGGCGATCTTAAAGAACGTGCTCATTGGAAGGACTATCGCAACGCTTACGAGGATATGTTGGCCAACACAAGCACTAAGCAGGCCCCGTGGTTCGTTATTCCCGCCGATGACAAGTGGTTTACACGCCTTTTGATCGCTGAGATCATTTGCACTGAATTAGAGAAGCTTAATCTTACTTTTCCTACGGTATCGTAA (SEQ ID NO: 35).

[0127] The test materials used in this invention are all ordinary commercially available products and can be purchased in the market.

[0128] The present invention will be further described below in conjunction with embodiments:

[0129] Example 1: Screening of aminopeptidase

[0130] The amino acid sequence (SEQ ID NO: 1) of the aminopeptidase Car01 from *Cystobasidium minutum* was input into the BLAST module of UniProt to retrieve 250 similar sequences. These sequences were then imported into MEGA software for alignment and a Neighbor-Joining phylogenetic tree was constructed. Ten sequences listed in Table 1 were selected from the phylogenetic tree for further validation.

[0131] Table 1

[0132]

[0133] The amino acid sequences of the above-mentioned candidate aminopeptidases were codon optimized using the online tool https: / / deepcodon.biodesign.ac.cn. The codon-optimized nucleotide sequence of aminopeptidase Car01 is shown in SEQ ID NO:2. The optimized nucleotide sequence was then used for whole-genome synthesis and constructed into the pET-28a vector, which was transformed into the BL21(DE3) expression host to prepare either stab-forming or glycerol-forming bacteria. The obtained stab-forming or glycerol-forming bacteria were streaked onto LB agar plates (Kan) and incubated overnight at 37°C. Then, three appropriately sized colonies were picked from each plate and transferred to a 10 mL tube of LB liquid medium (Kan), incubated at 37°C and 200 rpm for 6–8 h until turbidity was achieved. The remaining 9 mL of bacterial culture after preservation was transferred to 500 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 3–4 h until OD (dose dispersibility) was achieved. 600 =0.6~1.0, add 100µL of 0.5M IPTG (final concentration 0.1mM), and continue culturing at 20℃ and 200rpm for 16~20h.

[0134] Bacterial cells were collected by low-temperature centrifugation. After resuspending in disruption buffer (20mM K₂HPO₄ / KH₂PO₄, 100mM KCl, pH 7.6) at a ratio of 1g:9mL of wet cells, the cells were sonicated to obtain crude enzyme solution. The crude enzyme solution was then centrifuged at high speed, and the supernatant and precipitate were separated for protein electrophoresis to detect expression. Figure 1 and Figure 2 Car02, Car07, and Car10 were expressed in the precipitate, while the remaining aminopeptidases were partially expressed in the supernatant and completed autocleavage.

[0135] Weigh 2.02 g (100 mM, reaction volume 130 mL) of L-histidine and 1.82 g (100 mM) of β-alanine methyl hydrochloride, dissolve in 100 mL of pure water, adjust pH to 7.5, and bring the volume to 130 mL. Aliquot 10 mL into 50 mL centrifuge tubes, preheat at 30 °C, add 0.1 mL of the above crude enzyme solution, and take 100 µL samples at 15 min, 30 min, and 2 h respectively. After inactivation with 900 µL of 60% acetonitrile, the amount of carnosine (βAla-His) generated is detected by high performance liquid chromatography (HPLC). As shown in Table 2, the Car06 group had the highest product concentration, generating 27.35 mM after 2 h of reaction. The product concentrations of the Car03, Car05, and Car09 groups were lower than those of Car06 but higher than those of the other groups.

[0136] Table 2 Carnosine Concentration

[0137]

[0138] Example 2: Modification of aminopeptidase Car06

[0139] The AlphaFold predicted structure of aminopeptidase Car06 was analyzed, and the Cys85, Thr184, Lys262, Ser268, and Asn305 sites were selected. The primers listed in Table 3 were designed to construct plasmids of different mutants using the nucleotide sequence of Car06 (SEQ ID NO: 7) as a template.

[0140] Table 3

[0141]

[0142] The obtained plasmids were transformed into BL21(DE3) expression hosts and streaked onto LB agar plates (Kan) and incubated overnight at 37°C. Three appropriately sized colonies were then picked from each plate and transferred to 10 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 6–8 h until turbidity was reached. The remaining 9 mL of culture after preservation was transferred to 500 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 3–4 h until OD (Organic Discharge) was reached. 600 =0.6~1.0, add 100µL of 0.5M IPTG (final concentration 0.1mM), and continue culturing at 20℃ and 200rpm for 16~20h. Collect the bacterial cells by low-temperature centrifugation, and resuspend them in disruption buffer (20mM K2HPO4 / KH2PO4, 100mM KCl, pH7.6) at a ratio of 1g:9mL of wet bacterial cells. Then, sonicate to disrupt the cells and obtain the corresponding crude enzyme solution.

[0143] Weigh 2.02 g (100 mM, reaction volume 130 mL) of L-histidine and 1.82 g (100 mM) of β-alanine methyl hydrochloride, dissolve in 100 mL of pure water, adjust pH to 7.5, and bring the volume to 130 mL. Aliquot 10 mL into 50 mL centrifuge tubes, preheat at 30 °C, add 0.1 mL of the above crude enzyme solution, and take 100 µL samples at 15 min, 30 min, and 2 h respectively. After inactivation with 900 µL of 60% acetonitrile, the amount of carnosine (βAla-His) generated is detected by high performance liquid chromatography (HPLC). As shown in Table 4, the Car06_N305F group had the highest product concentration, generating 36.25 mM after 2 h of reaction, which is the optimal mutant; the N305G and Car06_N305V groups also showed increased product concentrations, while the Car06_S268C and Car06_N305P mutants resulted in loss of activity.

[0144] Table 4 Carnosine Concentration

[0145]

[0146] Example 3: Autocleavage optimization of aminopeptidase Car06_N305F

[0147] Car06_N305F glycerol bacteria were cultured to 2000 mL of TB liquid medium (Kan) and induced to express the enzyme at 20℃ with 0.1 mM IPTG. The bacterial cells were collected by low-temperature centrifugation, resuspended in disruption buffer at a ratio of 1 g:4 mL of wet cells, and then sonicated to obtain a crude enzyme solution. The crude enzyme solution was placed at 20℃, and samples were taken at 0 h, 4 h, and 24 h for protein electrophoresis to detect autocleavage. Figure 3 As the storage time increased, the ~44kD band gradually self-cleaved into ~29kD and ~15kD bands (not visible in protein electrophoresis), and self-cleavage was almost complete after 24 hours.

[0148] Weigh 1.55 g of L-histidine (100 mM, reaction volume 100 mL) and 1.67 g of β-alanine methyl hydrochloride (120 mM), dissolve in 80 mL of pure water, adjust pH to 7.5, and bring volume to 96 mL. After preheating the reaction solution to 20 °C, add 4 mL of crude enzyme solution that has been left to stand for 0 h. Take 100 µL samples at 1 h, 2 h, and 4 h, then inactivate with 900 µL of 60% acetonitrile and analyze the amount of carnosine (βAla-His) produced using high-performance liquid chromatography (HPLC). The next day, prepare the reaction solution in the same manner, preheat to 20 °C, add 4 mL of crude enzyme solution that has been left to stand for 24 h, and take 100 µL samples at 1 h, 2 h, and 4 h, then inactivate with 900 µL of 60% acetonitrile and analyze the amount of carnosine produced using high-performance liquid chromatography (HPLC). As shown in Table 5, the 24h group of self-cleaved aminopeptidase Car06_N305F exhibited higher carnosine synthesis performance.

[0149] Table 5

[0150]

[0151] Example 4: One-pot synthesis of acetyl tetrapeptide-5 (Ac-βAla-His-Ser-His)

[0152] The amino acid ligase Rsp1486 from Ralstonia solanacearum and the polyphosphoric acid kinase ChPPK2 (∆285-305) from Cytophaga hutchinsonii were selected and codons were optimized for Escherichia coli. The optimized nucleic acid sequence of Rsp1486 is shown in SEQ ID NO: 34, and the optimized nucleic acid sequence of ChPPK2 (∆285-305) is shown in SEQ ID NO: 35. The optimized nucleotide sequences were synthesized into a whole gene and constructed into the pET-28a vector, which was then transformed into the BL21(DE3) expression host to prepare stab-resistant bacteria or glycerol bacteria.

[0153] The obtained stab-forming bacteria or glycerol-containing bacteria were cultured to 2000 mL LB liquid medium (Kana) and induced to express the enzyme at 20℃ with 0.1 mM IPTG. The bacterial cells were collected by low-temperature centrifugation, resuspended in disruption buffer at a ratio of 1 g:4 mL wet cells, and then sonicated to obtain the corresponding crude enzyme solution. Protein electrophoresis was used to detect expression (see...). Figure 4 All of these are expressed in the Shangqing.

[0154] Weigh 31.03 g of L-histidine (217 mM, 920 mL reaction system), 14.52 g of N-acetyl-β-alanine methyl ester (109 mM), and 15.56 g of L-serine methyl ester hydrochloride (109 mM) and dissolve them in 750 mL of pure water. Adjust the pH to 7.5 and bring the volume to 900 mL. After preheating the reaction solution to 25 °C, add 20 mL of Car06_N305F crude enzyme solution (protein concentration 30.5 g / L, target enzyme content approximately 40%) that has been left to stand for 24 h. React for 4 h. High performance liquid chromatography (HPLC) detects the generation of 24.07 g of Ac-Carnosine (Ac-βAla-His) and 22.33 g of Ser-His dipeptide.

[0155] Add 15.91 g (26 mM) of sodium hexametaphosphate (corresponding to a reaction volume of 1 L), 8.13 g (40 mM) of magnesium chloride hexahydrate, and 0.69 g (2 mM) of adenosine monophosphate to the reaction solution from the first step. Dissolve these components, adjust the pH to 8.5, and bring the volume to 950 mL. After preheating at 37 °C, add 40 mL of Rsp1486 crude enzyme solution (protein concentration of 22.9 g / L, target enzyme content of approximately 35%) and 10 mL of ChPPK2 crude enzyme solution (total protein concentration of 26.2 g / L, target enzyme content of approximately 45%). React for 6 h. High-performance liquid chromatography (HPLC) detects the generation of 43.59 g (88.51 mM) of acetyl tetrapeptide-5. The reaction formula for the enzymatic synthesis of acetyl tetrapeptide-5 is shown below. Figure 5 .

[0156] Example 5: Sebaceous glands are an important component of the skin, primarily functioning to secrete sebum, which helps maintain skin moisture and barrier function. Under the stimulation of certain factors (such as hormones and the environment), sebaceous glands may over-secrete sebum, leading to oiliness, clogged pores, and skin problems such as acne. An in vitro oil-control experiment simulated the secretion process of sebaceous glands, studying the inhibitory effect of different ratios of acetyl tetrapeptide-5 and dipeptide-15 on sebum secretion by sebaceous gland cells. By quantitatively detecting the quality and changes of lipids secreted by cells, the oil-controlling ability of the raw materials was evaluated, providing a scientific basis for the development of oil-regulating products.

[0157] The raw materials and reagents used in the examples are all commercially available.

[0158] Among them, human sebaceous gland cells (SZ95) were obtained from Qingqi (Shanghai) Biotechnology Development Co., Ltd.; oleic acid was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.; DMEM medium was obtained from HyClone; serum was obtained from GibcoLife Technologies; penicillin-streptomycin bispecific antibody was obtained from GibcoLife Technologies; and lipid droplet red fluorescence detection kit was obtained from Shanghai Beyotime Biotechnology Co., Ltd. Experimental Procedure: 96-well black-plate (SZ95, P8) were seeded with 10,000 sebaceous gland cells per well. No substances were added to the control group. The positive control group received isotretinoin (VA, 100 μg / mL). Experimental group 1 received acetyl tetrapeptide-5 (T5, 10 μg / mL). Experimental group 2 received dipeptide-15 (D15, 500, 1000, 1500, 2000 μg / mL). Experimental group 3 received a mixture of acetyl tetrapeptide-5 (10 μg / mL) and dipeptide-15 (500, 1000, 1500, 2000 μg / mL) at different concentrations (1:50, 1:100, 1:150, 1:200). All cells were cultured for 24 hours. After washing once with PBS, the cells were stained with a lipid droplet red fluorescence detection kit for 20 minutes, washed twice with PBS, and the fluorescence intensity was measured (Ex: 552 nm, 636 nm). According to Yuan Shoujun's article "Mathematical Flaws in Several Commonly Used Drug Combination Calculation Methods and Solutions Using the One-Belt-One-Line Model" (Chinese Journal of Drug Vigilance, Vol. 18, No. 1, January 2021), the Bliss independent model is used to calculate the interaction of the compositions of different ratios in this invention, with the formula CI=E. A,B / (E A +E B -E A ×E B ), where E A E represents the effect of A when administered alone. B E represents the effect of drug B when administered alone. A,B The values ​​represent the combined effects of drugs A and B. CI < 1 indicates antagonistic effects, CI = 1 indicates additive effects, and CI > 1 indicates synergistic effects. Results are shown in Table 6 and... Figure 6 .

[0159] Table 6. Effects of different ratios of T5 and D15 on the inhibition of sebum secretion.

[0160]

[0161] Experimental results: as shown in Table 6 and Figure 6As can be seen, compared with the Control group, the lipid content decreased by 35.80% after the addition of 100 μg / mL isotretinoin to the positive control group, indicating that the experimental model was effective. Compared with the Control group, the lipid content decreased by 9.82% after the addition of 10 μg / mL acetyl tetrapeptide-5, and decreased by 8.38%, 10.14%, 13.59%, and 11.00% after the addition of 500, 1000, 1500, and 2000 μg / mL dipeptide-15, respectively. After the addition of 10 μg / mL acetyl tetrapeptide-5 and dipeptide-15 at concentrations of 500, 1000, 1500, and 2000 μg / mL, the lipid content decreased by 12.71%, 18.78%, 40.63%, and 28.93%, respectively, which were statistically significant (P<0.05). The results showed that when the mass ratio of acetyl tetrapeptide-5 to dipeptide-15 was 1:150 and 1:200, the CI was greater than 1, indicating that the compositions at both ratios had a synergistic effect on the inhibition of sebum secretion. Among them, the synergistic effect of 1:150 was the most obvious and the effect was significantly better than the compositions with a mass ratio of acetyl tetrapeptide-5 to dipeptide-15 of 1:50 or 1:100.

[0162] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for synthesizing acetyl tetrapeptide-5, characterized in that, Includes the following steps: S1. Under the catalysis of aminopeptidase or its mutant, N-acetyl-β-alanine methyl ester or its salt is reacted with L-histidine to obtain filamentous dipeptide and acetylcarnosine. S2. Add adenosine monophosphate, inorganic polyphosphate, polyphosphokinase and amino ligase to the reaction system of step S1, and generate acetyl tetrapeptide-5 through reaction.

2. The synthesis method according to claim 1, characterized in that, The aminopeptidase is derived from at least one of the following: Ajellomyces capsulatus, Colletotrichum gloeosporioides, Eutypa lata, Beauveria bassiana, Cyphellophoraeuropaea, Pyricularia oryzae, and Gaeumannomyces tritici. Preferably, the aminopeptidase comprises at least one of the following: (1) The enzyme Car02, derived from Ajellomyces capsulatus with UniProt ID accession number C6H1Y6; (2) Enzyme Car03 derived from Colletotrichum gloeosporioides with UniProt ID accession number T0M6U4; (3) Enzyme Car05, derived from Eutypa lata with UniProt ID accession number M7SYE6; (4) Enzyme Car06, derived from Beauveria bassiana with UniProt accession number J4UIY7; (5) Enzyme Car08 derived from Cyphellophora europaea with UniProt ID accession number W2S4G1; (6) Car09, an enzyme derived from Pyricularia oryzae with UniProt accession number G4NHJ4; (7) Car011, an enzyme derived from Gaeumannomyces tritici with UniProt ID accession number J3PFY1; The aminopeptidase mutant has any of the following amino acid sequences: (a) The amino acid sequence as shown in SEQ ID NO: 32; (b) An amino acid sequence obtained by substitution, deletion, addition or modification of one or more amino acids in the sequence shown in (a); (c) An amino acid sequence that is at least 80% identical to (a) or (b).

3. The synthesis method according to claim 1, characterized in that, The amino acid ligase is derived from Ralstonia solanacearum; the polyphosphoric acid kinase is derived from Cytophaga hutchinsonii.

4. The synthesis method according to claim 1, characterized in that, In step S1, the solvent for the reaction includes water; In step S2, the adenosine monophosphate compound includes adenosine monophosphate, adenosine triphosphate, or adenosine diphosphate; the polyphosphate includes sodium hexametaphosphate / or sodium tripolyphosphate; and the reaction includes reacting at 37°C for 6 hours.

5. The synthesis method according to claim 1, characterized in that, Step S1 further includes the following steps for preparing the aminopeptidase or a mutant thereof: The nucleic acid molecule encoding the aminopeptidase or its mutant is cloned into a vector, transformed into a prokaryotic expression system, and the prokaryotic expression system is cultured and induced to express. After separation and purification, a crude enzyme solution containing the aminopeptidase or its mutant is obtained.

6. A mutant of an aminopeptidase, characterized in that, It is a mutant of an amino acid enzyme with an amino acid sequence as shown in SEQ ID NO: 7, and the mutant includes at least one mutation site among C85S, C85A, T184V, K262A, N305G, N305V, and N305F. Preferably, the mutant has an amino acid sequence as shown in any of the following: (a) The amino acid sequence as shown in SEQ ID NO: 32; (b) An amino acid sequence obtained by substitution, deletion, addition or modification of one or more amino acids in the sequence shown in (a); (c) An amino acid sequence that is at least 80% identical to (a) or (b).

7. A biomaterial, characterized in that, Including any of the following: 1) Nucleic acids having sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13; 2) The nucleic acid encoding the mutant of claim 6; 3) An expression cassette containing the nucleic acid described in 1) or 2); 4) A recombinant vector comprising any one of the nucleic acids described in 1) to 2) or comprising the expression cassette described in 3); 5) Transfect or transform the host of the recombinant vector described in 4).

8. The biomaterial according to claim 6, characterized in that, The nucleic acid encoding the mutant of claim 6 has a sequence shown in any one of the following: (1) The nucleic acid sequence shown in SEQ ID NO: 33; (2) A nucleic acid sequence in which one or more bases are substituted, deleted, added, or modified in the sequence shown in (1); (3) A nucleic acid sequence that has at least 80% identity with the sequence shown in (1) or (2).

9. The biomaterial according to claim 6, characterized in that, The backbone of the recombinant vector includes pET series, pQE series, pHT series, pXMJ19, pHIL series, pPIC series, or pSET152 vectors; the host includes Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, or Streptomyces.

10. The use of the mutant of claim 5 or the biomaterial of any one of claims 6 to 8 in the biosynthesis of acetyl tetrapeptide.

11. A composition, characterized in that, Including acetyl tetrapeptide-5 and dipeptide-15; The mass ratio of the acetyl tetrapeptide-5 to the dipeptide-15 is 1:(150~200).

12. The composition according to claim 11, characterized in that, The mass ratio of the acetyl tetrapeptide-5 to the dipeptide-15 is 1:150 or 1:200.

Images