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CN-121975891-A - Method for biosynthesis of acetyl tetrapeptide-5, composition containing acetyl tetrapeptide and application thereof

CN121975891ACN 121975891 ACN121975891 ACN 121975891ACN-121975891-A

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 thereof. The invention uses low-cost natural amino acid as a substrate, uses specific aminopeptidase or mutants thereof as a biocatalyst, combines amino acid ligase and polyphosphatase, and can efficiently synthesize acetyl tetrapeptide-5 through one-step enzymatic reaction. Compared with the traditional chemical synthesis method, the method has the advantages of low raw material cost, no use of organic solvent, mild reaction conditions, high regioselectivity, environmental friendliness, low production cost and the like. The invention also provides a composition comprising acetyl tetrapeptide-5 and dipeptide-15, which has an inhibition effect on grease secretion remarkably superior to that of the independent components and has a synergistic oil control effect under a specific proportion.

Inventors

  • ZHANG ZHANG
  • HAO WEI
  • LIAO LIZHI
  • DENG PENGHUA
  • CHEN XUELIAN
  • FANG XIN
  • HE SUIPING

Assignees

  • 深圳瑞德林生物技术有限公司

Dates

Publication Date
20260505
Application Date
20260206

Claims (12)

  1. 1. The synthesis method of the acetyl tetrapeptide-5 is characterized by comprising the following steps: s1, under the catalysis of aminopeptidase or mutants thereof, N-acetyl-beta-alanine methyl ester or salts thereof reacts with L-histidine to obtain silk group dipeptide and acetyl carnosine; S2, adding an adenosine phosphate compound, inorganic polyphosphate, polyphosphate kinase and amino ligase into the reaction system in the step S1, and reacting to generate the acetyl tetrapeptide-5.
  2. 2. The synthetic method of claim 1, wherein the source of aminopeptidase comprises at least one of yarrowia capsulata Ajellomyces capsulatus, colletotrichum gloeosporioides Colletotrichum gloeosporioides, botrytis cinerea Eutypa lata, beauveria bassiana Beauveria bassiana, fucus vesiculosus Cyphellophora europaea, pyriform Pyricularia oryzae, and hologra tritici Gaeumannomyces tritici; preferably, the aminopeptidase comprises at least one of: (1) Enzyme Car02 with UniProt ID accession number C6H1Y6, derived from Ajellomyces capsulatus; (2) Enzyme Car03 with UniProt ID accession number T0M6U4, derived from Colletotrichum gloeosporioides; (3) Enzyme Car05, uniProt ID accession number M7SYE6, derived from Eutypa lata; (4) Enzyme Car06 with UniProt ID accession No. J4UIY7, derived from Beauveria bassiana; (5) Enzyme Car08 with UniProt ID accession number W2S4G1, derived from Cyphellophora europaea; (6) Enzyme Car09 with UniProt ID accession No. G4NHJ4, derived from Pyricularia oryzae; (7) Enzyme Car011 with UniProt ID accession J3PFY1, derived from Gaeumannomyces tritici; the aminopeptidase mutant has an amino acid sequence as set forth in any one of: (a) An amino acid sequence shown as SEQ ID NO. 32; (b) An amino acid sequence obtained by substituting, deleting, adding or modifying one or more amino acids in the sequence shown in (a); (c) An amino acid sequence having at least 80% identity to (a) or (b).
  3. 3. The method of claim 1, wherein the source of amino acid ligase comprises Ralstonia solanacearum and the source of polyphosphate kinase comprises Cytophaga hutchinsonii.
  4. 4. The synthetic method according to claim 1, wherein in step S1, the solvent of the reaction comprises water; in the step S2, the adenosine phosphate compound comprises adenosine monophosphate, adenosine triphosphate or adenosine diphosphate, the polyphosphate comprises sodium hexametaphosphate and/or sodium tripolyphosphate, and the reaction comprises 37 ℃ reaction for 6 hours.
  5. 5. The synthetic method of claim 1, wherein step S1 further comprises the step of preparing the aminopeptidase or mutant thereof as follows: cloning the nucleic acid molecule encoding the aminopeptidase or the mutant thereof to a vector, then converting the nucleic acid molecule into a prokaryotic expression system, culturing and inducing the prokaryotic expression system, and separating and purifying to obtain crude enzyme liquid containing the aminopeptidase or the mutant thereof.
  6. 6. A mutant of aminopeptidase, which is characterized by being a mutant of an aminopeptidase having an amino acid sequence as shown in SEQ ID No. 7, said mutant comprising at least one mutation site in C85S, C85A, T184V, K262A, N305G, N305V, N F; Preferably, the mutant has an amino acid sequence as set forth in any one of the following: (a) An amino acid sequence shown as SEQ ID NO. 32; (b) An amino acid sequence obtained by substituting, deleting, adding or modifying one or more amino acids in the sequence shown in (a); (c) An amino acid sequence having at least 80% identity to (a) or (b).
  7. 7. A biomaterial characterized by comprising any one of the following: 1) Nucleic acid with the sequence shown as 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) A nucleic acid encoding the mutant of claim 6; 3) An expression cassette comprising the nucleic acid of 1) or 2); 4) A recombinant vector comprising any one of 1) to 2) or 3) the expression cassette; 5) Transfecting or transforming the host of the recombinant vector of 4).
  8. 8. The biomaterial of claim 6, wherein the nucleic acid encoding the mutant of claim 6 has a sequence as set forth in any one of: (1) A nucleic acid sequence shown in SEQ ID NO. 33; (2) A nucleic acid sequence in which one or more bases or is substituted, deleted, added or modified in the sequence shown in (1); (3) A nucleic acid sequence having at least 80% identity to the sequence set forth in (1) or (2).
  9. 9. The biomaterial according to claim 6, wherein the backbone of the recombinant vector comprises pET series, pQE series, pHT series, pXMJ, pHIL series, pPIC series or pSET152 vector, and the host comprises E.coli, B.subtilis, C.glutamicum, pichia pastoris or Streptomyces.
  10. 10. Use of the mutant according to claim 5 or the biomaterial according to any one of claims 6 to 8 in biosynthesis of acetyl tetrapeptides.
  11. 11. A composition comprising acetyl tetrapeptide-5 and dipeptide-15; The mass ratio of the acetyl tetrapeptide-5 to the dipeptide-15 is 1 (150-200).
  12. 12. The composition of claim 11, wherein the mass ratio of the acetyl tetrapeptide-5 and the dipeptide-15 is 1:150 or 1:200.

Description

Method for biosynthesis of acetyl tetrapeptide-5, composition containing acetyl tetrapeptide and application thereof Technical Field 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 thereof. Background Sebum is produced primarily by sebaceous glands, and synthesis is regulated by multiple factors such as endocrine (androgens, etc.), metabolic enzymes (e.g., fatty acid synthase FASN), local inflammation, neuro-endocrine signaling, and the skin microbiota. Current oil control strategies are generally directed to physical adsorption of excess sebum, inhibition of biochemical pathways of sebum synthesis, regulation of keratinous/pore conditions, or inhibition of sebum-related inflammatory/microbial activity 1. Excessive sebum secretion can lead to skin barrier dysfunction, pore blockage and greasy feel, increase skin inflammation and risk of acne formation, and promote overgrowth of microorganisms (e.g., propionibacterium acnes), exacerbating acne, comedones and skin sensitivity problems. The long-term grease surplus can also cause the thickening of the horny layer, so that the problems of rough skin, uneven luster, easiness in cosmetic removal and the like are caused, meanwhile, the oxidative stress of the skin is accelerated, the inflammation and early aging are induced, and the overall skin health and appearance are affected. Chinese patent CN119656057a discloses a new application of acetyl tetrapeptide-5 in the preparation of oil-controlling cosmetics. Cell experiments prove that the composition can obviously reduce the lipid content of human sebaceous gland cells (SZ 95), has the highest inhibition rate of 87 percent and has obvious oil control effect. Patent CN101061135A discloses a method for solid phase synthesis of acetyl tetrapeptide-5, which comprises condensing Fmoc-L-His (Trt) -OH to 2-chlorotrityl resin, sequentially deprotecting and coupling 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, and deprotecting and cutting to obtain acetyl tetrapeptide-5 (βala-His-Ser-His). The raw materials such as the 2-chlorotrityl resin and the amino acid with side chain protection used in the method are expensive, and the condensation coupling is required to be completed in a large amount of organic solvents such as DMF, so that the production cost is increased, the environment is possibly polluted, a large amount of reagents are required to be consumed in the subsequent cutting and purifying processes, the process is complex, the yield is low, and the requirement of large-scale industrial production is difficult to meet. Therefore, the development of an efficient, green and low-cost biosynthesis method for preparing the acetyl tetrapeptide has important significance in promoting the wide application of the acetyl tetrapeptide in the fields of cosmetics, medicines and the like. U.S. patent No. 20230320967a discloses a novel use of the proline containing dipeptide compound, dipeptide diammine Ding Xianbian amine diacetate, in regulating sebaceous gland function and controlling oil. It can be used as DPP4 inhibitor, and has effects in inhibiting sebaceous cell proliferation, reducing neutral lipid generation, and remarkably improving oily light, pores and acne. Clinical researches show that the sebum secretion and skin glossiness can be effectively reduced after the composition is continuously used for 28 days, and the composition has remarkable oil control and skin beautifying effects. Chinese patent CN116744896a discloses a skin care composition containing palmitoyl dipeptide-7 (Pal-KT) and acetyl tetrapeptide-11 (Ac-PPYL). The two are used for up-regulating PPARA and MSMO gene expression together in a ratio of 10:1 to 1:10, improving cellular ATP level, promoting cellular energy metabolism and ECM repair, improving skin aging problems such as wrinkles, looseness and barrier damage, and the like, and the effect is obvious and the low dose is effective. Chinese patent CN1886114B discloses a series of specific amino acid derivatives (including dipeptide-15) and salts thereof, which significantly inhibit keratosis, shrink pores and improve rough skin, thereby forming keratosis inhibitor, pore shrinking agent and external composition using the same as main active ingredient, and are widely applied in skin care preparations for face and body. The composition has the advantages of single acting target point of acetyl tetrapeptide-5, limited sebum-related inflammation or microorganism regulation by inhibiting lipid generation, long clinical service period of proline dipeptide derivative, insignificant short-term effect, no multipass synergistic effect for DPP4, main improvement of aging and barrier by combination of palmitoyl dipeptide-7 and acetyl tetrapeptide-11, insufficient oil c