Preparation method of acetyl hexapeptide-8, subtilisin variant and application of subtilisin variant

Abstract

The invention provides a preparation method of acetyl hexapeptide-8, a subtilisin variant and application of the subtilisin variant, and relates to the technical field of cosmetics. According to the preparation method of the acetyl hexapeptide-8, disclosed by the invention, Ac-Glu-Glu-Met-Gln-OCam-Leu and Arg-Arg-NH2 hydrochloride are added into water, and the acetyl hexapeptide-8 is obtained under the action of a subtilisin variant; wherein the subtilisin variant is one or more of enzyme with an amino acid sequence as shown in SEQ ID NO.1 and specific variants of the enzyme. The preparation method is carried out in water under the action of the specific subtilisin variant, so that toxic organic solvent residues are avoided; the raw material conversion rate is high, the concentration of a target product in the reacted material is improved, and the difficulty of subsequent purification operation is further reduced; the raw materials used in the invention reduce the use of protected amino acids, and compared with the existing solid phase method, the raw material cost can be effectively reduced, so that the method is an efficient, environment-friendly and economic acetyl hexapeptide-8 preparation method, and has wide application prospects and industrial potential.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a preparation method of acetyl hexapeptide-8, a subtilisin variant and its application. Background Art

[0002] The formation of dynamic wrinkles (expression lines) is the result of repeated contractions of facial expression muscles that mechanically pull on the skin surface. Its generation mechanism involves the dual effects of muscle dynamics and skin structure. When the expression muscles contract, the dermis layer has a weakened resilience due to the loss of collagen and elastic fibers; at the same time, the epidermis layer is more prone to form temporary wrinkles due to a decrease in water content, which will solidify into static wrinkles in the long term. In this process, the release of acetylcholine neurotransmitter at the neuromuscular junction plays a key role. The action potential of motor neurons triggers the influx of calcium ions, which promotes the release of acetylcholine by synaptic vesicles through the SNARE protein complex, and then activates muscle cell contraction.

[0003] Based on this physiological mechanism, acetyl hexapeptide-8 (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2) can competitively inhibit the assembly of the SNARE complex by mimicking the C-terminal sequence (Glu-Glu-Met-Gln-Arg-Arg) of the SNAP-25 protein, thereby reducing the release amount of acetylcholine and gently inhibiting excessive muscle contraction. Its acetylated molecular structure enhances the enzymatic stability, and finally realizes the targeted intervention of dynamic wrinkles. This biomimetic blocking strategy has both reversibility and safety, and has become the core technical solution in the anti-wrinkle field to replace botulinum toxin. By targeting the formation mechanism of dynamic wrinkles, acetyl hexapeptide-8 effectively reduces the excessive contraction of facial expression muscles, thereby delaying the transformation of dynamic wrinkles into static wrinkles and realizing the optimization of the anti-wrinkle effect.

[0004] At present, chemical methods such as solid-phase synthesis and liquid-phase synthesis are the mainstream processes for synthesizing acetyl hexapeptide-8. For example, Patent CN114057838A discloses a method for solid-phase synthesizing acetyl hexapeptide-8. In this method, Fmoc-Arg(Pbf)-OH is condensed onto an amino resin, and then Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Glu(OtBu)-OH are deprotected and coupled in sequence to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-amino resin, which is then deprotected, acetylated, and cleaved to obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. Other patents for the solid-phase synthesis method of acetyl hexapeptide-8 include Patent CN111620928A, Patent CN103694316A, Patent CN102603869A, etc. Another example is that Patent CN113968895A discloses an intermediate composition of acetyl hexapeptide-8, its preparation method and application. This method is a liquid-phase synthesis method, which specifically includes first synthesizing Ac-Glu(OtBu)-Glu(OtBu)-Met-OH and H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-NH2 separately, and then condensing and cleaving to obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. Other patents for the liquid-phase synthesis method of acetyl hexapeptide-8 include Patent CN116987148A, CN113845586A, and CN103613642A.

[0005] In the above preparation methods of acetyl hexapeptide-8, raw materials such as amino resin or 2-chlorotrityl resin used in the solid-phase synthesis method are all expensive, thus restricting the industrial production of acetyl hexapeptide-8; while the liquid-phase synthesis method uses side-chain protected amino acids as substrates, with relatively high raw material costs, and the condensation needs to be completed in a large amount of organic solvents such as DMF. Therefore, subsequent purification operations for reducing solvent residues are required, which increases the overall process cost and is not conducive to large-scale production. Summary of the Invention

[0006] Aiming at the deficiencies in the prior art, the present invention discloses a preparation method of acetyl hexapeptide-8, a subtilisin variant and its application. This preparation method is carried out in water, solving the problems of high cost and unfavorable large-scale production in the prior solid-phase or liquid-phase methods for preparing acetyl hexapeptide-8.

[0007] To achieve the above technical objectives, on the one hand, the present invention provides a method for preparing acetyl hexapeptide-8. In this method, Ac-Glu-Glu-Met-Gln-OCam-Leu and Arg-Arg-NH2 hydrochloride are reacted in water under the action of a Bacillus subtilis protease variant to obtain the acetyl hexapeptide-8;

[0008] Among them, the Bacillus subtilis protease variant is one or more of the following enzymes:

[0009] The enzyme with the amino acid sequence shown in SEQ ID NO.1, the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101C mutation, the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101C and P129A mutations, the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101C and P129N mutations, the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101A mutation, the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101A and P129A mutations, and the enzyme with the amino acid sequence in which the amino acids shown in SEQ ID NO.1 undergo S101A and P129N mutations.

[0010] The reaction process of this preparation method is as follows:

[0011]

[0012] The method for preparing acetyl hexapeptide-8 of the present invention is carried out in an aqueous phase under the action of a specific Bacillus subtilis protease variant. Compared with traditional methods, it avoids the use and residue of toxic organic solvents, thus significantly reducing the difficulty of post-treatment and improving the feasibility and safety of operation; in addition, the preparation method of the present invention has a high raw material conversion rate and can effectively increase the concentration of the target product in the reaction material. This feature not only improves production efficiency but also further reduces the difficulty of subsequent purification operations, making it more suitable for large-scale industrial production; in terms of cost, the technical solution of the present invention reduces the use of protected amino acids. Compared with the existing solid-phase synthesis method, this method can effectively reduce raw material costs, thus showing more significant advantages in terms of economy and market competitiveness.

[0013] In an alternative example of the present invention, the nucleotide sequence of the enzyme with the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.2 (codon-optimized).

[0014] In an alternative example of the present invention, the nucleotide sequence of the upstream primer for constructing the Sbt70-S101C mutant of the enzyme having the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.4.

[0015] In an alternative example of the present invention, the nucleotide sequence of the upstream primer for constructing the Sbt70-S101A mutant of the enzyme having the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.6.

[0016] In an alternative example of the present invention, the nucleotide sequence of the upstream primer for constructing the Sbt70-P129A mutant of the enzyme having the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8.

[0017] In an alternative example of the present invention, the nucleotide sequence of the upstream primer for constructing the Sbt70-P129N mutant of the enzyme having the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.10.

[0018] Furthermore, the types of the subtilisin variants were explored and optimized. Based on the exploration experiments, optionally, the subtilisin variant is an enzyme shown by the amino acid sequence in which the amino acids shown in SEQ ID NO.1 have S101C and P129A mutations, an enzyme shown by the amino acid sequence in which the amino acids shown in SEQ ID NO.1 have S101C and P129N mutations, an enzyme shown by the amino acid sequence in which the amino acids shown in SEQ ID NO.1 have S101A and P129A mutations, or an enzyme shown by the amino acid sequence in which the amino acids shown in SEQ ID NO.1 have S101A and P129N mutations. Further optionally, the subtilisin variant is an enzyme shown by the amino acid sequence in which the amino acids shown in SEQ ID NO.1 have S101C and P129N mutations. The amino acid sequence of this subtilisin variant is as shown in SEQ ID NO.11, and its nucleotide sequence is as shown in SEQ ID NO.12.

[0019] Furthermore, the method for preparing acetyl hexapeptide-8 of the present invention comprises the following steps:

[0020] (1) Add a buffer reagent, Ac-Glu-Glu-Met-Gln-OCam-Leu, and Arg-Arg-NH2 hydrochloride to water, dissolve and adjust the pH value to obtain a first material;

[0021] (2) Add the Bacillus subtilis protease variant to the first material, and react to obtain the acetyl hexapeptide-8.

[0022] Furthermore, the pH value of the first material is 7.5 to 10.0, preferably 8.5 to 9.5, and more preferably the pH value is 8.5. In actual operation, it is optional to adjust the pH value of the mixed material in step (1) and make up the volume to 1000 ml for subsequent operations.

[0023] Furthermore, in the step (2), the mass ratio of the Bacillus subtilis protease variant to the substrate Ac-Glu-Glu-Met-Gln-OCam-Leu is 1:(0.5 to 2), preferably 1:0.75 to 1:1.875.

[0024] Furthermore, the reaction temperature in the step (2) is 20 to 30 °C, preferably 25 to 30 °C, and more preferably 25 °C; the reaction time is 1.5 h to 3 h, preferably 2 h. The operation conditions of the preparation method of acetyl hexapeptide-8 in the present invention are mild, and the raw material conversion rate is high, which is suitable for wide promotion and use.

[0025] Furthermore, the buffer reagent includes at least one of tris(hydroxymethyl)methylglycine, glycine, dipotassium hydrogen phosphate, and disodium hydrogen phosphate.

[0026] Furthermore, the molar concentration of the buffer reagent is 20 mM to 100 mM.

[0027] Furthermore, the preparation method of the Arg-Arg-NH2 hydrochloride includes: S1, L-Arg (L-arginine) is synthesized to obtain Arg-Arg under the action of an amino acid ligase and a polyphosphate kinase; the reaction process is as follows:

[0028]

[0029] S2, the Arg-Arg is obtained to Arg-Arg-NH2 under the action of an amidase and a polyphosphate kinase; the reaction process is as follows:

[0030]

[0031] S3, the reaction solution containing Arg-Arg-NH2 is ultrafiltered to remove proteins, nanofiltrated and concentrated to remove salts, adjusted to pH for crystallization, and dried to obtain Arg-Arg-NH2 hydrochloride.

[0032] Furthermore, the amino acid sequence of the amino acid ligase is as shown in SEQ ID NO.13, and its nucleotide sequence is as shown in SEQ ID NO.14 (codon-optimized).

[0033] Furthermore, the polyphosphate kinase is derived from Sulfurovum lithotrophicum, and its amino acid sequence is shown in SEQ ID NO.15, and its nucleotide sequence is shown in SEQ ID NO.16 (codon-optimized).

[0034] Furthermore, the amidase is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.17, and its nucleotide sequence is shown in SEQ ID NO.18 (codon-optimized).

[0035] Furthermore, steps S1 to S2 are carried out in one pot, thereby simplifying the preparation process of Arg-Arg-NH2 hydrochloride; the raw material used in this preparation process is natural Arg (arginine) that does not require protection, with lower cost; in addition, the examples of the present invention confirm that the concentration of Arg-Arg can reach nearly 90 mM, with a high raw material conversion rate and important practical application value.

[0036] On the other hand, the present invention provides a subtilisin variant, which is an enzyme having the amino acid sequence shown in SEQ ID NO.1, an enzyme having the amino acid sequence in which the amino acid at position S101 of SEQ ID NO.1 is mutated to C, an enzyme having the amino acid sequence in which the amino acids at positions S101 and P129 of SEQ ID NO.1 are mutated to C and A respectively, an enzyme having the amino acid sequence in which the amino acids at positions S101 and P129 of SEQ ID NO.1 are mutated to C and N respectively, an enzyme having the amino acid sequence in which the amino acid at position S101 of SEQ ID NO.1 is mutated to A, an enzyme having the amino acid sequence in which the amino acids at positions S101 and P129 of SEQ ID NO.1 are mutated to A and A respectively, and an enzyme having the amino acid sequence in which the amino acids at positions S101 and P129 of SEQ ID NO.1 are mutated to A and N respectively.

[0037] On the other hand, the present invention provides the use of the above-mentioned subtilisin variant in the preparation of acetyl hexapeptide-8.

[0038] The amino acid sequences involved in the present invention include:

[0039] SEQ ID NO.1:

[0040] AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIVSTLPGNKYGAKSGTAMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVEAAAQ

[0041] SEQ ID NO.11:

[0042] AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVAGGASFVPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGCGQYSWIINGIEWAIANNMDVINMSLGGNSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIVSTLPGNKYGAKSGTAMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVEAAAQ

[0043] SEQ ID NO.13:

[0044] MLRILLTNSDKPEPIQFFQKDKETNDSINISVTTESCYAPLYSHWADHVYIVDDVTDLTVMKSLMLEILKVGPFDHIVSTTEKSILTGGFLRSYFGIAGPGFETALYMTNKLAMKTKLKMEGIPVADFLCVSQVEDIPAAGEKLGWPIIVKPALGSGALNTFIIHSLDHYEDLYSTSGGLGELKKNNSLMIAEKCIEMEEFHCDTLYADGEILFVSISKYTVPLLKGMAKIQGSFILSQNDPVYAEILELQKSVAQAFRITDGPGHLEIYRTHSGELIVGEIAMRIGGGGISRMIEKKFNISLWESSLNISVYRDPNLTVNPIEGTVGYFSLPCRNGTIKEFTPIEEWEKLAGILEVELLYQEGDVVDEKQSSSFDLARLYFCLENENEVQHLLALVKQTYYLHLTEDHMMNQ

[0045] SEQ ID NO.15:

[0046] MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDITKKEQKKRLEARKTDPLKQWKLSPIDAKAQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAP

[0047] SEQ ID NO.17:

[0048] MSKGTTSQDAPFGTLLGYAPGGVAIYSSDYSSLDPQEYEDDAVFRSYIDDEYMGHKWQCVEFARRFLFLNYGVVFTDVGMAWEIFSLRFLREVVNDNILPLQAFPNGSPRAPVAGALLIWDKGGEFKDTGHVAIITQLHGNKVRIAEQNVIHSPLPQGQQWTRELEMVVENGCYTLKDTFDDTTILGWMIQTEDTEYSLPQPEIAGELLKISGARLENKGQFDGKWLDEKDPLQNAYVQANGQVINQDPYHYYTITESAEQELIKATNELHLMYLHATDKVLKDDNLLALFDIPKILWPRLRLSWQRRRHHMITGRMDFCMDERGLKVYEYNADSASCHTEAGLILERWAEQGYKGNGFNPAEGLINELAGAWKHSRARPFVHIMQDKDIEENYHAQFMEQALHQAGFETRILRGLDELGWDAAGQLIDGEGRLVNCVWKTWAWETAFDQIREVSDREFAAVPIRTGHPQNEVRLIDVLLRPEVLVFEPLWTVIPGNKAILPILWSLFPHHRYLLDTDFTVNDELVKTGYAVKPIAGRCGSNIDLVSHHEEVLDKTSGKFAEQKNIYQQLWCLPKVDGKYIQVCTFTVGGNYGGTCLRGDESLVIKKESDIEPLIVVKK

[0049] The nucleic acid molecule sequences involved in the present invention include:

[0050] SEQ ID NO.2:

[0051] GCCCAGTCCGTCCCCTACGGTGTGTCGCAAATTAAGGCACCCGCATTGCACTCTCAGGGGTACACCGGAAGCAACGTCAAGGTAGCAGTGATCGACAGCGGGATCGATAGCTCTCACCCGGATTTGAATGTCGCTGGCGGCGCATCATTCGTGCCGAGTGAAACCAACCCATTCCAGGATAACAATTCACATGGGACGCACGTCGCGGGTACTGTACTTGCCGTCGCGCCATCCGCGAGCTTGTACGCCGTAAAAGTCCTGGGAGCTGACGGTAGCGGTCAATACAGTTGGATCATCAACGGAATTGAGTGGGCGATTGCCAACAATATGGATGTCATCAACATGTCGCTGGGAGGCCCTAGCGGGTCCGCCGCTTTGAAAGCCGCAGTGGACAAAGCGGTCGCCTCGGGCGTTGTCGTAGTGGCGGCGGCTGGTAATGAAGGCACAAGTGGTAGTTCAAGTACGGTC GGATATCCGGGAAAGTACCCAAGTGTGATTGCAGTAGGGGCCGTGGACTCCTCGAACCAACGTGCTTCCTTTAGCTCGGTTGGCCCAGAATTGGATGTTATGGCACCCGGCGTGAGCATTGTCTCAACACTGCCCGGAAACAAATATGGAGCAAAAAGTGGTACTGCAATGGCTTCACCTCACGTTGCGGGAGCAGCCGCACTTATCCTGTCTAAGCATCCCAACTGGACCAATACCCAGGTACGCAGTTCCCTTGAGAATACCACCACCAAACTGGGAGACTCTTTTTATTATGGAAAGGGCTTAATCAACGTAGAGGCTGCAGCGCAGTAA

[0052] SEQ ID NO.3:

[0053] GGAGCTGACGGTTGCGGTCAATACAG

[0054] SEQ ID NO.4:

[0055] TGTATTGACCGCAACCGTCAGCTCCC

[0056] SEQ ID NO.5:

[0057] TGGGAGCTGACGGTGCCGGTCAATACAGTTG

[0058] SEQ ID NO.6:

[0059] AACTGTATTGACCGGCACCGTCAGCTCCCAG

[0060] SEQ ID NO.7:

[0061] TCGCTGGGAGGCGCTAGCGGGTCCGC

[0062] SEQ ID NO.8:

[0063] CGGACCCGCTAGCGCCTCCCAGCGAC

[0064] SEQ ID NO.9:

[0065] TCGCTGGGAGGCAATAGCGGGTCCGCC

[0066] SEQ ID NO.10:

[0067] GCGGACCCGCTATTGCCTCCCAGCGAC

[0068] SEQ ID NO.12:

[0069] GCCCAGTCCGTCCCCTACGGTGTGTCGCAAATTAAGGCACCCGCATTGCACTCTCAGGGGTACACCGGAAGCAACGTCAAGGTAGCAGTGATCGACAGCGGGATCGATAGCTCTCACCCGGATTTGAATGTCGCTGGCGGCGCATCATTCGTGCCGAGTGAAACCAACCCATTCCAGGATAACAATTCACATGGGACGCACGTCGCGGGTACTGTACTTGCCGTCGCGCCATCCGCGAGCTTGTACGCCGTAAAAGTCCTGGGAGCTGACGGTTGCGGTCAATACAGTTGGATCATCAACGGAATTGAGTGGGCGATTGCCAACAATATGGATGTCATCAACATGTCGCTGGGAGGCAATAGCGGGTCCGCCGCTTTGAAAGCCGCAGTGGACAAAGCGGTCGCCTCGGGCGTTGTCGTAGTGGCGGCGGCTGGTAATGAAGGCACAAGTGGTAGTTCAAGTACGGTCGGATATCCGGGAAAGTACCCAAGTGTGATTGCAGTAGGGGCCGTGGACTCCTCGAACCAACGTGCTTCCTTTAGCTCGGTTGGCCCAGAATTGGATGTTATGGCACCCGGCGTGAGCATTGTCTCAACACTGCCCGGAAACAAATATGGAGCAAAAAGTGGTACTGCAATGGCTTCAC CTCACGTTGCGGGAGCAGCCGCACTTATCCTGTCTAAGCATCCCAACTGGACCAATACCCAGGTACGCAGTTCCCTTGAGAATACCACCACCAAACTGGGAGACTCTTTTTATTATGGAAAGGGCTTAATCAACGTAGAGGCTGCAGCGCAGTAA

[0070] SEQ ID NO.14:

[0071]

[0072] SEQ ID NO.16:

[0073] ATGAAGAAAAACATCTACAAGAAAGAGTTGTATAAACTTCAGGTGGAGCTGGTTAAGTTCCAGAAGTATGTCATCGAGGAGAACGTTGCCGTCTGCTTGGTTCTGGAGGGTCGTGACACTGCAGGCAAGGATGGAACTATTAAACGCTTCACAGAACACTTGAGCCCCCGCGAAGCCCGCACCGTAGCATTAGGGGTGCCTAGTGATAAAGAAAAAAAGAGCTGGTATTTTCAGCGCTATGTACCCCACCTTCCTTCGGCGGGGGAAATTGTATTCTTCAATCGTTCTTGGTATAACCGTGCGGGAGTAGAAAAAGTCATGGGGTTCTGTACAAAAAAACAGTATAAAGCGTTCATGGAGGAGGTCGGTTCCTTTGAACAAATGTTGACGCACTCCAATATTCGCTTTTTCAAATACTACTTGGATATTACGAAAAAGGAGCAAAAAAAACGTCTGGAGGCACGTAAGACCGATCCCCTTAAGCAGTGGAAACTGTCCCCTATCGACGCGAAAGCACAAAAAATGTGG GATGCTTATTCCAAGGCGCGTGATGACATGTTCAACAAGACGTCCTTCATCTACGCTCCTTGGTACGTTGTCCATACCGACGATAAAAAGGAAGCACGTATTAACATTATGAAGCATTTCCTTTCGTTGAACGACTATCCCGACAAAGACAAAGCGTTATTGGTTTATGACCACGACGTCATCTGCAAATTCGACCCTGTTTGTTACGAGAAAGAGATGATTGCACCCTAA

[0074] SEQ ID NO.18:

[0075]

[0076] Compared with the prior art, the beneficial effects of the present invention are as follows: The preparation method of acetyl hexapeptide-8 of the present invention is carried out in water under the action of a specific subtilisin variant, avoiding the use and residue of toxic organic solvents, reducing the difficulty of post-treatment, and having strong operability; the raw material conversion rate is high, increasing the concentration of the target product in the reaction material, further reducing the difficulty of subsequent purification operations, and being suitable for large-scale production; in addition, the raw materials used in the above technical solution reduce the use of protected amino acids, and can effectively reduce the raw material cost compared with the existing solid-phase method. It is an efficient, environmentally friendly and economical preparation method of acetyl hexapeptide-8, with broad application prospects and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0078] Figure 1 Shows the reaction result of amino acid ligase catalyzed synthesis of Arg-Arg in Example 1 of the present invention;

[0079] Figure 2 Shows the molecular docking model of subtilisin variant Sbt70 and acetyl hexapeptide-8 in Example 3;

[0080] Figure 3 Shows the comparison chart of different subtilisin variants catalyzing the generation of acetyl hexapeptide-8 in Example 3;

[0081] Figure 4 Shows the comparison chart of Argireligase immobilized enzyme generating acetyl hexapeptide-8 under different pH conditions in Example 4;

[0082] Figure 5 Shows the comparison chart of Argireligase immobilized enzyme generating acetyl hexapeptide-8 under different temperature conditions in Example 4;

[0083] Figure 6 Shows the result of subtilisin variant Argireligase catalyzing the preparation of acetyl hexapeptide-8 in Example 4;

[0084] Figure 7 Shows the liquid chromatography result of the reaction material in Example 4;

[0085] Figure 8 Shows the first-order mass spectrometry result of the acetyl hexapeptide-8 prepared in Example 4;

[0086] Figure 9Show the SDS-PAGE electrophoresis diagram of the expression and purification of the subtilisin variant Argireligase in Example 4. Detailed implementation mode

[0087] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only for more detailed description and should not be construed as limiting the present invention in any form, that is, it is not intended to limit the protection scope of the present invention.

[0088] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0089] Example 1 Synthesis of Arg-Arg under the action of amino acid ligase

[0090] The optimized nucleotide sequences were obtained by inputting the amino acid sequences of the modified amino acid ligase BsRizA-I7T_I33T_R35E and the polyphosphate kinase SlPPK2 from Sulfurovum lithotrophicum into the codon optimization website https: / / sg.idtdna.com / CodonOpt, and then submitted to Tsingke Biotechnology Co., Ltd. for whole gene synthesis and constructed on the pET-28a vector to obtain the expression plasmids pET28a-BsRizA-I7T_I33T_R35E and pET28a-SlPPK2.

[0091] The obtained plasmids were transformed into BL21(DE3) competent cells, and then induced to express for 16-20 h at 20 °C under the condition of 0.1 mM IPTG in LB medium (Kan). The cells were collected by centrifugation, resuspended in the lysis buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6) according to the ratio of 1 g of wet cells to 4 mL, and then sonicated to obtain the crude enzyme solution (it should be noted that in the embodiments of the present invention, the crude enzyme solutions of other enzymes not mentioned here were obtained by operating according to this method).

[0092] Weigh 34.84 g (200 mM) of L-arginine, 33.65 g (55 mM) of sodium hexametaphosphate, 12.20 g (60 mM) of magnesium chloride hexahydrate, and 1.10 g (2 mM) of adenosine triphosphate disodium salt, dissolve them in 800 mL of pure water, adjust the pH to 9.0 with ammonia water and make up the volume to 940 mL. After preheating at 38 °C, add 50 mL of the crude enzyme solution of BsRizA-I7T_I33T_R35E (amino acid ligase) and 10 mL of the crude enzyme solution of SlPPK2 (polyphosphate kinase), and start the reaction. Samples are taken during the reaction process, and the production of Arg-Arg in the product is detected by high performance liquid chromatography (HPLC). As Figure 1 shown, 88.2 mM of Arg-Arg is produced after 4 h of reaction, and the content of Arg-Arg in the reaction material reaches nearly 90 mM after the reaction, with a high raw material conversion rate.

[0093] Example 2 Synthesis of Arg-Arg-NH2 under the action of amidase

[0094] Select the amidase EcGspS derived from Escherichia coli, perform whole gene synthesis after codon optimization, and construct it on the pET-28a vector to obtain the expression plasmid pET28a-EcGspS. The plasmid is transformed into BL21(DE3) competent cells, and then induced to express for 4 - 6 h at 37 °C under the condition of 0.1 mM IPTG in LB medium (Kan). The bacterial cells are collected by centrifugation, resuspended according to 1 g of wet bacterial cells: 4 mL of lysis buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6), and then ultrasonically lysed to obtain the crude enzyme solution.

[0095] Adjust the pH of the reaction solution obtained in Example 1 to 7.0 and make up the volume to 1050 mL. Add 50 mL of the crude enzyme solution of EcGspS. After reacting at 38 °C for 1 h, all Arg-Arg is converted into Arg-Arg-NH2. After removing proteins by ultrafiltration, concentrating and desalting by nanofiltration, adjusting the pH for crystallization, and drying, 26.32 g of Arg-Arg-NH2 hydrochloride is obtained.

[0096] Example 3 Substrate adaptability modification of subtilisin variant Sbt70

[0097] In this example, the substrate adaptability modification of subtilisin variant Sbt70 (amino acid sequence shown in SEQ ID NO.1) includes the following steps:

[0098] First, perform whole gene synthesis on the optimized nucleotide sequence of subtilisin variant Sbt70 and construct it on the pET-28a vector to obtain the expression plasmid pET28a-Sbt70.

[0099] Using subtilisin Sbt70 (PDB: 1SUA) as the target and Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 as the ligand, molecular docking was performed using AutoDock Vina (as Figure 2 shown). Using the primers shown in Table 1, different mutant plasmids were constructed with pET28a-Sbt70 as the template, and the serial numbers of the mutation sites were referred to the annotations in the Sbt70 crystal structure.

[0100] Table 1

[0101] Mutant primer Primer sequence S101C-up GGAGCTGACGGTTGCGGTCAATACAG S101C-dn TGTATTGACCGCAACCGTCAGCTCCC S101A-up TGGGAGCTGACGGTGCCGGTCAATACAGTTG S101A-dn AACTGTATTGACCGGCACCGTCAGCTCCCAG P129A-up TCGCTGGGAGGCGCTAGCGGGTCCGC P129A-dn CGGACCCGCTAGCGCCTCCCAGCGAC P129N-up TCGCTGGGAGGCAATAGCGGGTCCGCC P129N-dn GCGGACCCGCTATTGCCTCCCAGCGAC

[0102] Subsequently, the obtained plasmid was transformed into BL21(DE3) competent cells, and then two-stage expanded culture was carried out in LB medium (Kan) and induced to express for 16 h under the conditions of 20 °C and 0.1 mM IPTG. The bacterial cells were collected by centrifugation, resuspended in lysis buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6) at a ratio of 1 g of wet bacterial cells to 4 mL, and then sonicated; after high-speed centrifugation, 100 g / L of LX-1000 IDA-Ni resin (Lanxiao Technology) was added to the supernatant, and the immobilized enzyme was collected after incubation for 2 h.

[0103] Weighed 0.627 g (50 mM) of Tricine, 0.052 g (1 mM) of Ac-Glu-Glu-Met-Gln-OCam-Leu, and 0.031 g (1 mM) of Arg-Arg-NH2 hydrochloride and dissolved them in 60 mL of pure water, adjusted the pH to 8.8 and made up the volume to 70 mL. Aliquoted into 7 portions, and 0.1 g of subtilisin variants of different mutants were added respectively, reacted at 25 °C for 30 min, and the formation of the product Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 was detected by liquid chromatography-mass spectrometry (LC-MS). The test results are as Figure 3 shown.

[0104] Combined Figure 3Based on the results shown, it is speculated that subtilisin variant Sbt70 has relatively strong hydrolysis ability, so the content of acetyl hexapeptide-8 in the material after its catalytic reaction is relatively low; the mutation of Ser at position 101 to Ala or Cys both endows certain peptide synthesis ability, and the reaction conversion rates of mutants S101A and S101C reach about 11% and 20% respectively; further superimposing the mutation of Pro at position 129 to Ala or Asn may expand the substrate pocket and further increase the product concentration, and the reaction conversion rates of mutants S101A_P129A, S101A_P129N, and S101C_P129A reach about 48%, 56%, and 65% respectively. By comparison, the preferred subtilisin mutant is S101C_P129N. Under its catalysis, 0.82 mM of product can be generated after reacting for 30 min (in this example, this mutant is named Argireligase for easy elaboration in subsequent examples).

[0105] Based on this example, the subtilisin variant in the method for preparing acetyl hexapeptide-8 of the present invention can be selected from one or more of the following enzymes: the enzyme with the amino acid sequence shown in SEQ ID NO.1, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C mutation, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C and P129A mutations, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C and P129N mutations, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101A mutation, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101A and P129A mutations, and the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101A and P129N mutations. Further, it can be selected from the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C and P129A mutations, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C and P129N mutations, the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101A and P129A mutations, or the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101A and P129N mutations. More preferably, it is the enzyme with the amino acid sequence in which the amino acid shown in SEQ ID NO.1 undergoes S101C and P129N mutations.

[0106] Example 4 Method for synthesizing acetyl hexapeptide-8 using subtilisin variant Argireligase

[0107] 1. Exploration and optimization of reaction pH

[0108] Weigh 0.717 g (50 mM) of Tricine, 0.060 g (1 mM) of Ac-Glu-Glu-Met-Gln-OCam-Leu, and 0.035 g (1 mM) of Arg-Arg-NH2 hydrochloride, dissolve them in 65 mL of pure water, divide them into 8 portions, adjust the pH to 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 respectively, and make up the volume to 10 mL. Then add 0.1 g of immobilized Argireligase to each portion and react at 25 °C for 30 min. Use liquid chromatography-mass spectrometry (LC-MS) to detect the formation of the product Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2.

[0109] The results are as Figure 4 shown. The immobilized Argireligase has synthetic activity under alkaline conditions with pH 7.5 - 10.0. When the pH is 8.5 - 9.5, the product concentration is relatively high; when the pH value is 8.5, the product concentration reaches the highest value of 0.90 mM.

[0110] 2. Exploration and optimization of reaction temperature

[0111] Weigh 0.538 g (50 mM) of Tricine, 0.045 g (1 mM) of Ac-Glu-Glu-Met-Gln-OCam-Leu, and 0.026 g (1 mM) of Arg-Arg-NH2 hydrochloride, dissolve them in 50 mL of pure water, adjust the pH to 8.5, and make up the volume to 60 mL. Divide them into 6 portions, preheat them at 4 °C, 20 °C, 25 °C, 30 °C, 38 °C, and 45 °C respectively, then add 0.1 g of immobilized Argireligase to each portion and react for 30 min. Use liquid chromatography-mass spectrometry (LC-MS) to detect the formation of the product Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2.

[0112] The results are as Figure 5 shown. The immobilized Argireligase can react at 20 - 45 °C. Preferably, the product concentration is relatively high when reacting at 20 - 30 °C, and the further preferred reaction temperature is 25 °C.

[0113] 3. Laboratory scale-up

[0114] Weigh 8.96 g (50 mM) of Tricine, 74.88 g (100 mM) of Ac-Glu-Glu-Met-Gln-OCam-Leu, and 87.76 g (200 mM) of Arg-Arg-NH2 hydrochloride into 900 mL of pure water, adjust the pH to 8.5, make up the volume to 1000 mL, add 40 g of immobilized Argireligase, and react at 25 °C.

[0115] As Figure 6 shown, 98 mM of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 is formed after 2 h of reaction. After ultrafiltration, nanofiltration, crystallization, and drying, 59.24 g of acetyl hexapeptide-8 is obtained. As Figure 7 shown, the purity of the sample is 99.59%; as Figure 8 shown, the quasi-molecular ion peak at 889.7500 in the positive mode of the first-order mass spectrum of the sample is [M+H] + , and 445.2500 is [M+2H] 2+ , which is consistent with the result calculated by ChemDraw. The acetyl hexapeptide-8 prepared by the present invention has high purity, good quality, and no organic solvent residue. When it is applied to the preparation of cosmetics, it can reduce the excessive contraction of facial muscles, thereby delaying the transformation of dynamic wrinkles into static wrinkles and achieving the anti-wrinkle effect.

[0116] In addition, this example also shows the preparation and purification of the subtilisin variant Argireligase:

[0117] Take the glycerol bacteria of Argireligase BL21(DE3) and expand them in LB liquid medium (Kan) respectively, and then inoculate them for small tank fermentation. The formula of the fermentation medium is shown in Table 2.

[0118] Table 2

[0119]

[0120] When the biomass OD600 = 40 - 60, start cooling to 20 °C. At this time, take the uninduced cell sample, and then add 0.1 mM IPTG to induce protein expression for 20 - 24 h. Harvest the cells when OD600 = 160 - 240. Centrifuge to collect the cells and store them at -20 °C for later use. Resuspend the wet cells at a ratio of 1 g of wet cells : 4 mL of lysis buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6), and then disrupt the cells by high-pressure homogenization. After high-speed centrifugation, add 100 g / L of LX-1000 IDA-Ni resin (Lanxiao Technology) to the supernatant, incubate for 2 h, and then collect the immobilized enzyme. Take 2 g of the immobilized enzyme and add 4 mL of elution buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, 250 mM imidazole, pH 7.6), incubate for 10 min and then take a sample; after detection by protein electrophoresis, Argireligase is expressed in the supernatant and is well adsorbed on the LX-1000 IDA-Ni resin (as Figure 9 shown).

[0121] It should be noted that the above content is a further detailed description of the present invention in combination with specific embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple improvements can be made, and all of them should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for preparing acetyl hexapeptide-8, characterized in that, The acetyl hexapeptide-8 is obtained by reacting Ac-Glu-Glu-Met-Gln-OCam-Leu with Arg-Arg-NH2 hydrochloride in water under the action of a subtilisin variant. Among them, the subtilisin variant is one or more of the following enzymes: The enzyme having the amino acid sequence shown in SEQ ID NO.1, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has an S101C mutation, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101C and P129A mutations, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101C and P129N mutations, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has an S101A mutation, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101A and P129A mutations, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101A and P129N mutations.

2. The preparation method of acetyl hexapeptide-8 according to claim 1, characterized in that The subtilisin variant is the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101C and P129A mutations, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101C and P129N mutations, the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101A and P129A mutations, or the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101A and P129N mutations.

3. The preparation method of acetyl hexapeptide-8 according to claim 2, wherein The subtilisin variant is the enzyme having the amino acid sequence in which the amino acid shown in SEQ ID NO.1 has S101C and P129N mutations.

4. The preparation method of acetyl hexapeptide-8 according to claim 1, characterized in that, It includes the following steps: (1) Add a buffer reagent, Ac-Glu-Glu-Met-Gln-OCam-Leu, and Arg-Arg-NH2 hydrochloride to water, and adjust the pH value after dissolution to obtain a first material. (2) Add the subtilisin variant to the first material and react to obtain the acetyl hexapeptide-8.

5. The method for preparing acetyl hexapeptide-8 according to claim 4, wherein The pH value of the first material is 7.5 to 10.0, preferably 8.5 to 9.5, and more preferably 8.

5.

6. The method for preparing acetyl hexapeptide-8 according to claim 4, wherein In the step (2), the mass ratio of the subtilisin variant to the substrate Ac-Glu-Glu-Met-Gln-OCam-Leu is 1: (0.5 to 2), preferably 1:(0.75 to 1:1.875); and / or, the reaction temperature in the step (2) is 20 to 45 °C, preferably 20 to 30 °C, and more preferably 25 °C; The reaction time is 1.5 h to 3 h, preferably 2 h.

7. The method for preparing acetyl hexapeptide-8 according to claim 4, wherein The buffer reagent includes at least one of tris(hydroxymethyl)methylglycine, glycine, dipotassium hydrogen phosphate, and disodium hydrogen phosphate; and / or, the molar concentration of the buffer reagent is 20 to 100 mM.

8. The preparation method of acetyl hexapeptide-8 according to any one of claims 1 to 7, characterized in that, The preparation method of the Arg-Arg-NH2 hydrochloride includes: S1. L-Arg is reacted under the action of an amino acid ligase and a polyphosphate kinase to obtain Arg-Arg; S2. The Arg-Arg is reacted under the action of an amidase and a polyphosphate kinase to obtain Arg-Arg-NH2; S3. The reaction solution containing Arg-Arg-NH2 is subjected to ultrafiltration to remove proteins, nanofiltration and concentration to remove salts, pH adjustment for crystallization, and drying to obtain the Arg-Arg-NH2 hydrochloride; Preferably, steps S1 to S2 are carried out in one pot.

9. A subtilisin variant, characterized in that, The subtilisin protease variant is an enzyme having the amino acid sequence shown in SEQ ID NO.1, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with an S101C mutation, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with S101C and P129A mutations, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with S101C and P129N mutations, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with an S101A mutation, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with S101A and P129A mutations, an enzyme having the amino acid sequence shown by the amino acid sequence of SEQ ID NO.1 with S101A and P129N mutations.

10. Use of the subtilisin protease variant according to claim 9 in the preparation of acetyl hexapeptide-8.

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