Enzyme composition and its use in the preparation of acetyl hexapeptide-8

Abstract

The present application relates to the field of biotechnology, and particularly to an enzyme composition and its application in the preparation of acetyl hexapeptide-8. The present application provides an enzymatic preparation process of acetyl hexapeptide-8, which uses Glu-Glu-Met and Gln-Arg-Arg as substrates, constructs a hexapeptide skeleton under the catalysis of ATP-dependent polypeptide ligase, then completes acetylation modification by N-terminal acetylation enzyme with the participation of acetyl CoA, and realizes C-terminal amidation by amination enzyme. The preparation process can be implemented in segments or synthesized in one step, and continuous production can also be realized by using an immobilized enzyme reactor; the preparation process is used for the synthesis of acetyl hexapeptide-8, has core advantages such as high economy, strong process integration, product purity > 99.5%, and the like, and provides a new industrialization paradigm for the green manufacturing of anti-wrinkle peptide raw materials.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to enzyme compositions and their use in the preparation of acetyl hexapeptide-8. Background Technology

[0002] Currently, the preparation methods for acetyl hexapeptide-8 on the market mainly focus on chemical methods, such as solid-phase synthesis, liquid-phase synthesis, and segmented condensation strategies. For example, Lipotec uses solid-phase synthesis technology to form the target polypeptide chain and complete the amino-terminal modification by stepwise linking the acetyl-glutamic acid-glutamic acid-methionine-glutamine-arginine-arginine sequence. Shandong Jitai uses a seven-step liquid-phase synthesis method to sequentially synthesize key intermediates such as Boc-Met-Gln-OH and Ac-Glu(OtBu)-Glu(OtBu)-OH, and finally obtains acetyl hexapeptide-8 through recrystallization purification. Hantai Bio uses a segmented condensation strategy to synthesize three dipeptide monomers in the liquid phase: Ac-Glu(O-tBu)-Glu(O-tBu)-OH, H-Met-Gln(Trt)-OH, and H-Arg(Pbf)-Arg(Pbf)-NH2, and then complete the directional linking synthesis of acetyl hexapeptide-8 through the DCC / HOBt system.

[0003] However, existing methods for synthesizing acetyl hexapeptide-8 cannot achieve precise sequence assembly, resulting in peptide chain mismatch. Furthermore, traditional solid-phase synthesis involves numerous byproducts such as the removal of protecting groups, leading to low purity and complex synthesis steps. The yield also needs further improvement. Therefore, it is necessary to develop new routes for high-purity acetyl hexapeptide-8. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an enzyme composition and its application in the preparation of acetyl hexapeptide-8.

[0005] This invention provides a polypeptide ligase variant, HexPLigase, whose amino acid sequence is shown in SEQ ID NO:1.

[0006] This invention provides a combinatorial enzyme, which includes combinatorial enzyme 1 and / or combinatorial enzyme 2;

[0007] The combined enzyme 1 includes: the polypeptide ligase variant HexPLigase, acetyltransferase or its variant NkAcetylTrans, and amination enzyme or its variant CpAmid as described in this invention;

[0008] The combined enzyme 2 includes: the polypeptide ligase variant HexPLigase, acetyltransferase or its variant NkAcetylTrans, acetyl-CoA synthase or its variant PfAcCoASyn, ATP regenerase or its variant PsPPK, and amination enzyme or its variant CpAmid.

[0009] The Uniprot ID number of the acetyltransferase is A0A918XJD1;

[0010] The Uniprot ID number of the acetyl-CoA synthase is G0EH45;

[0011] The Uniprot ID number of the ATP regenerator is A0A1D7QNW6;

[0012] The Uniprot ID number of the ammoniata enzyme is A0A1D7QNW6.

[0013] Furthermore, in the combined enzyme described in this invention,

[0014] The amino acid sequence of the variant of the acetyltransferase is shown in SEQ ID NO:2;

[0015] The amino acid sequence of the variant of the acetyl-CoA synthase is shown in SEQ ID NO:3;

[0016] The amino acid sequence of the ATP regenerase variant is shown in SEQ ID NO:4;

[0017] The amino acid sequence of the amination enzyme variant is shown in SEQ ID NO:5.

[0018] This invention provides a biomaterial comprising at least one of the following: (I) to (V)

[0019] I) Nucleic acid encoding the polypeptide ligase variant HexPLigase or the combination enzyme of the present invention;

[0020] II) Expression units containing nucleic acids as shown in I);

[0021] III) Recombinant vectors containing nucleic acids as shown in I) or expression units as shown in II);

[0022] IV) Integrating the genome into the nucleic acid shown in I) or the expression unit shown in II), or transfecting or transforming the host cell with the recombinant vector shown in III);

[0023] V) Cultures obtained by culturing host cells as shown in IV)

[0024] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0025] In this invention, the nucleic acid may be optimized or unoptimized. These optimizations include, but are not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0026] The present invention also provides an expression unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, etc.

[0027] The present invention provides a recombinant vector comprising at least one of the nucleic acids or expression units described in the present invention and a vector backbone.

[0028] Furthermore, the vector backbone of the present invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and the present invention does not limit this. The animals include mammals and non-mammals, and the mammalian expression vectors include, but are not limited to, pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, and pColdII. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, and pAUR23.

[0029] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in various prokaryotic expression vectors. For example, its backbone vector may be pET28a.

[0030] This invention provides host cells for transforming or transfecting the recombinant vector. The source of the host cells includes plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention is not limited thereto. In a specific embodiment of this invention, the host cells are derived from bacteria; specifically, *Escherichia coli*, and more specifically, *E. coli* (BL21) or *E. coli* DH5α.

[0031] The transformation methods include chemical transformation and electroconversion; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, and lentivirus transfection.

[0032] The present invention provides an immobilized enzyme comprising at least one of the ligase variants or combination enzymes described in the present invention and a vector;

[0033] Furthermore, the carrier includes epoxy resin, more specifically LX-1000 EP epoxy resin.

[0034] The present invention provides a composition comprising at least one of the combined enzymes or immobilized enzymes described in the present invention and a reactant;

[0035] The reactant 1 includes reactant 1 and reactant 2;

[0036] The reactant 1 includes reactant 1 and reactant 2;

[0037] The reactant 1 includes: Glu-Glu-Met, Gln-Arg-Arg, magnesium chloride, ATP, acetyl-CoA, CpAmid, and ammonium chloride;

[0038] The reactant 2 includes: Glu-Glu-Met, Gln-Arg-Arg, ATP, acetyl-CoA, magnesium chloride, acetic acid, sodium hexametaphosphate, and ammonium chloride.

[0039] This invention provides the use of at least one of the following (i) to (v) in the preparation of acetyl hexapeptide-8:

[0040] i) The polypeptide ligase variant described in this invention;

[0041] ii) The combined enzyme described in this invention;

[0042] iii) The biomaterials described in this invention;

[0043] iv) The immobilized enzyme described in this invention;

[0044] v) The composition described in this invention.

[0045] This invention provides a product containing acetyl hexapeptide-8, characterized in that the raw materials used in its production include at least one of the following: a) to e)

[0046] a) The polypeptide ligase variant described in this invention;

[0047] b) The combined enzyme described in this invention;

[0048] c) The biomaterials described in this invention;

[0049] d) The immobilized enzyme described in this invention;

[0050] e) The composition described in this invention.

[0051] This invention provides a method for preparing acetyl hexapeptide-8, characterized in that it involves producing acetyl hexapeptide-8 using at least one of the following methods: A) to E).

[0052] A) The polypeptide ligase variant described in this invention;

[0053] B) The combined enzyme described in this invention;

[0054] C) The biomaterials described in this invention;

[0055] D) The immobilized enzyme described in this invention;

[0056] E), The composition described in this invention.

[0057] Furthermore, the preparation method of the present invention includes the following steps:

[0058] Acetyl hexapeptide-8 was synthesized using Glu-Glu-Met and Gln-Arg-Arg as substrates, with the participation of combinatorial enzyme 1 in the combinatorial enzyme described in this invention and reactant 1 in the composition described in this invention; or

[0059] Acetyl hexapeptide-8 was synthesized using Glu-Glu-Met and Gln-Arg-Arg as substrates, with the participation of combinatorial enzyme 2 in the combinatorial enzyme described in this invention and reactant 2 in the composition described in this invention.

[0060] In this invention, the method for preparing acetyl hexapeptide-8 can be described as follows: acetyl hexapeptide-8 can be synthesized in one step from substrates Glu-Glu-Met and Gln-Arg-Arg with the participation of the above-mentioned combined enzymes and reactants, or acetyl hexapeptide-8 can be synthesized through multiple steps. This invention does not limit the specific steps.

[0061] Specifically, the combined enzymes can be directly mixed or immobilized enzymes formed through immobilization; in the immobilized enzyme system, the polypeptide ligase (HexPLigase), acetyltransferase (NkAcetylTrans), acetyl-CoA synthase (PfAcCoASyn), ATP regenerator (PsPPK), and aminotransferase (CpAmid) are mixed and immobilized in a unit activity ratio of (1.5~2.5):(2.5~3.5):(2.5~3.5):(1.5~2.5):(1.5~2.5); specifically, a unit activity ratio of 2:3:3:2:2 followed by mixing and immobilization yields the best results;

[0062] In the preparation method described in this invention, acetyl hexapeptide-8 can be synthesized through a non-ATP regeneration system and an acetyl-CoA regeneration system with the participation of combined enzyme 1 and reactant 1; acetyl hexapeptide-8 can also be synthesized through an ATP regeneration system and an acetyl-CoA regeneration system with the participation of combined enzyme 2 and reactant 2.

[0063] Experimental results show that the above methods can all achieve the synthesis of acetyl hexapeptide-8 with high purity and low by-products; however, the final yield is significantly improved when using enzyme variants compared to wild-type enzymes. Furthermore, the one-step synthesis of acetyl hexapeptide-8 offers advantages of higher purity and higher yield while being simpler in its steps. Combined with immobilized enzymes, production costs are further reduced, allowing for multiple uses of the enzymes.

[0064] This invention provides a highly efficient and precise enzymatic process for the preparation of acetyl hexapeptide-8. Using two tripeptide products, Glu-Glu-Met and Gln-Arg-Arg, as raw materials, a hexapeptide backbone is constructed under the catalysis of an ATP-dependent polypeptide ligase (HexPLigase). Subsequently, acetylation modification is achieved through N-terminal acetylation enzyme (NkAcetylTrans) with the participation of acetyl-CoA, followed by C-terminal amidation by an aminotransferase (CpAmid). This process can be implemented in stages or continuously using an immobilized enzyme reactor. Addressing the high cost of ATP and acetyl-CoA, an innovative ATP regeneration enzyme (PsPPK)-driven ATP regeneration system and an acetyl-CoA synthase (PfAcCoASyn) coupled acetyl-CoA cyclic regeneration system were developed, reducing cofactor consumption by 85%. The directed evolution-modified HexPLigase exhibits over 20-fold increased catalytic efficiency and reduces byproduct formation to below 0.5%. With an optimized activity ratio (2:3:2), the three-enzyme system can achieve a single-tank conversion rate of over 80%. This technology boasts core advantages such as high atom economy, strong process integration, and product purity >99.5%, providing a new industrialization paradigm for the green manufacturing of anti-wrinkle peptide raw materials.

[0065] The preparation method of the multi-enzyme complex system used in this patent is different from the chemical synthesis methods commonly used in the market today. For the first time, it uses inexpensive and readily available raw materials such as Glu-Glu-Met, Gln-Arg-Arg, magnesium chloride hexahydrate, acetic acid, and sodium hexametaphosphate as initial raw materials. It utilizes an independently developed multi-enzyme system to obtain acetyl hexapeptide-8 with high yield and high quality through splicing and modification, providing a new approach to the preparation of acetyl hexapeptide-8 using this multi-enzyme complex system.

[0066] This invention provides an enzymatic preparation process for acetyl hexapeptide-8. After enzyme optimization, the process uses Glu-Glu-Met and Gln-Arg-Arg as substrates to construct a hexapeptide backbone under the catalysis of an ATP-dependent polypeptide ligase. Subsequently, acetylation modification is completed by an N-terminal acetyltransferase with the participation of acetyl-CoA, and C-terminal amidation is achieved by an aminotransferase. This preparation process can be implemented in stages or in one step, and can also be used in an immobilized enzyme reactor for continuous production. This process for the synthesis of acetyl hexapeptide-8 has key advantages such as high economic efficiency, strong process integration, and product purity >99.5%, providing a new industrial paradigm for the green manufacturing of anti-wrinkle peptide raw materials. Attached Figure Description

[0067] Figure 1 The image shows an SDS-PAGE gel image of the purified enzyme, where M: protein standard marker; 1 is HexPLigase; 2 is NkAcetylTrans; 3 is PfAcCoASyn; 4 is PsPPK; and 5 is CpAmid.

[0068] Figure 2 HPLC chromatogram of purified acetyl hexapeptide-8. Detection conditions: Column: Waters XBridge-C18 (4.6 mm × 250 mm, 5 μM); Mobile phase: acetonitrile + 0.1% phosphoric acid aqueous solution, flow rate 1.0 mL / min, 30 °C. o C, detected at 215nm;

[0069] Figure 3 Mass spectrum of acetyl hexapeptide-8, Agilent Ultivo LC / TQ. Detailed Implementation

[0070] This invention provides an enzyme composition and its application in the preparation of acetyl hexapeptide-8. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. 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. 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.

[0071] HexPLigase: Derived from Saccharothrix australisiensis (Uniprot ID: A0A495VZ10), this natural enzyme (WTHexPLigase) has low activity against its substrate. After systematic modification, its activity and expression level were improved. The specific mutation sites are: W22T, G122M, H123F, L131V, S132N, Q133A, K231R, D248W, P284S, M305K, R306G, and E325R.

[0072] Acetyltrans: Derived from Nocardiopsis kunsanensis (Unipert ID: A0A918XJD1) in Gunsan City, this natural enzyme (WTNkAcetylTrans) has certain activity against the substrate. After systematic modification (NkAcetylTrans), its activity and stability have been improved. The specific mutation sites are: I18V, D22Q, R65S, V89I, D176N, V203K, D204C, F238L, E239D and S336A.

[0073] Acetyl-CoA synthase (PfAcCoASyn): Derived from Pyrolobus fumarii (Uniprot ID: G0EH45), this natural enzyme (WTPfCoASyn) has a high regeneration capacity of acetyl-CoA, but its expression level is relatively poor. By modifying the structure of this enzyme (PfAcCoASyn), its activity and expression are significantly improved. The mutation sites are: H36T, Y123I, R208P, F209Q, R286N, and N428S.

[0074] ATP regenerase (PsPPK): Derived from Pedobacter steynii (Uniprot ID: A0A1D7QNW6), this natural enzyme (WTPsPPK) has good ATP regeneration activity and expression level, but its stability is not ideal. After site-directed mutagenesis at several sites (PsPPK), its stability has been improved significantly. The specific mutation sites are: M67L, Q89E, C133Y and S174T.

[0075] Aminotransferase (CpAmid): Derived from the sea ice psychrophilic bacterium Colwellia sp. (Uniprot ID: A0A1D8RSI1), this natural enzyme (WTCpAmid) has low substrate activity and low expression level. Through systematic experimental optimization and modification, a mutant enzyme (CpAmid) with improved performance in all aspects was finally obtained. The specific mutation sites are: D9V, W120T, D121H, Q159L, N172S, W189M, E206Q, F279D, N303K, E304R, D339N, S423L and L424F.

[0076] Amino acid sequence of HexPLigase: MVQLLVGHDFNEDLRNRNAAATYSQRLTWFARDGDVLVLPTEPDKAYVEYVTGWTGTSAESLRFVVPPPGEGAIGRLTRERLRDPEFLDRLRAAAEDRVIDHVYALWPDARVVELADALGVMFAVPGRGFVNAGGDAVANSKALFRVIARGVGVPVAEGAVCLHPRAAEQAVTALLDAGHPAMLKNEFMSGGWGNEIISRTPHIDPIGARRLVVVADAITLRDYLEEHWDRLTGDGRHSLVVERYHPWSSAAFAEFHVGDDGVRFGGQGQLLSLPQAASVIPASNVDAGRMAEIIDGGRRLGEAKGAIGYRGVLSADAIVTPAGRVLFTEYNGRSTGSTHLYGVVGEQVIGPGYAEDRFIVERIGTPPWSVTTFAEAVDRLAGTDLGYDRASRRGVLFVHAFNPAANCVPYCVVAESMDAATEVEQRLGELFDFAPPIA (SEQ ID NO:1);

[0077] Amino acid sequence of NkAcetylTrans: MSEQTPVPAGFRGLAGNVGIKQHHDDFFVVASEVPAHVSAVFTRSRFAGPSVVRSRDAAADGNVSGVVTLARNANVATGTEGEAHAREIQELAASAAGVPADEMIVASTGVIGRPYPMDRIRSTFHALSSPLPAADLDRSAAAMMTTDTRPKTASARVGGATVTGIAKGVGMIEPNMATMLAWFFTDAEIDRSVLDAVFRRVKCRTFNALSIDSDTSTSDSAAIFANGLAGPVGTDALDEALYGIALQLVRMIASDGEGAGKLIEVRATGARDDAQAKRVAKTVVNSPLVKTAVHGADPNWGRVAMAVGKCSDETDIHPENVRIVFGDVETYPEPADEETLRRAAEHMAGDEVVIGVDLGIADGSFTVYGCDLTEGYIRINADYTT (SEQ ID NO:2);

[0078] Amino acid sequence of PfAcCoASyn: MAVVGASRNPRKVGHIILRNILEYGFRGRVYPVNPTASTILGLRAYPSLTSLPETVDVAVVAVPAEKVPRVVQDAGEAGIPFLVIVSSGFREVGRHDLEEEVLRIARKYGVRIIGPNVAGIVITPARLNATFGPRDVIPGSIAFISQSGAFAIALMGATINEGMGVSAIVSVGNKVDIDDVDLLEYFETDSNTSVVLMYVEGLRDGRPQLRVASKVSLRKPVIIIKAGRTEAGAKAAASHTGSLAGSFDVYRAAFRQSGVLLATSMEEAFDAAKAFAWNPLPRGDNVLVITNGGGAGIQAVDTLVERRIRVEEPPTELQDRMKSFLPNFASTRNPIDLTGMAHADWFYRAIKEAMRHPWVDAIVVLYTQTGLSGPVETAKAILDAIREEGHAKPVTVGLLGGPECIRAARLLTKERVAAYPTPERAASAMSFLVEYVRLRDYVKERLSELQYA (SEQ ID NO:3);

[0079] Amino acid sequence of PsPPK: MKKEIEKYLAVPGKKVLLKDHQTSYNGDQEKEDGKEEMDEVKERLSKLQETLYAANSHSILILFQALDAAGKDSAISHVMSGLNPQGCEVYSFKAPTSEEYEHDFLWRHYKALPERGRIGIHNRSHYENVLVYKVHPEYVLNENIPGYQDLKLIDDKFWKKRYESIRSFEQHITENGITIIKIFLNVSKEEQKSRFLDRINDPAKNWKFSSSDITERGRWDEYMKAYETAIEETSTEQAPWYIIPADKKWHARLAISQILEEHFNRLDLKFPVLAEEEAKKLDEIKELLLKE (SEQ ID NO:4);

[0080] Amino acid sequence of CpAmid: MTKIKNIQVVSFGTPIGISMGNVTAYSSDYDTVDPLIYKSRSHFRSYVDDIYMGHKWQCVEFARRWLYTNKGYIFNDVAMAYEIFNLRSVRDIVNNSELALNAFENGAKRLPEVGSLLITHEGGEFEETGHVAIITAVFNDKVHIAEQNMAFAPWPNGLNYSREIKAKLGSSGDYWLHCPSDGSTILGMLIQTDDETDALISSLPQPAVFNIEACNAPAQNKANTPWLNLANDDEYAFFQMMAGHRLSCDDASALRYYRISPQAHLALETATDELHQMDMHATDYVLEHPELLEKFGLPKNLKRKIKRSWSNRANQLVTSRFDFSMSEHGLKVYEYNCNSASCYMEVGKVQGKWLKHFQVKGGTDAGTALFSHLVKAWQARAIDSVIHILQDDDPEETYHALFMKNAIEAAGHQCKILVGIDLFTCNKNNEIEDSAGIKIQWIWKTWAWETALDEIRANQDDVINKAQHNKSSKPSLSDVLLNDNIMVFEPLWTLIPSNKAILPVLCMLFPNHPYLLNTSFELTDELKQTGYVSKPIVGRCGANIKIIDENKQVIAEKPGNFETRDQIYQTLFPLPYVENKYVQVCTFTANGNYAGSGVRVDSSMIIDKDSDCLALQCKHDV (SEQ ID NO:5);

[0081]

[0082]

[0083]

[0084] Nucleotide sequence of PsPPK: atgaaaaaagaaattgaaaaatatctggcggtgccgggcaaaaaagtgctgctgaaagatcatcagaccagctataacggcgatcaggaaaaagaagatggcaaagaagaaatggatgaagtgaaagaacgcctgagcaaactgcaggaaaccctgtatgcggcgaacagccatagcattctgattctgtttcaggcgctggatgcggcgggcaaagatagcgcgattagccatgtgatgagcggcctgaacccgcagggctgcgaagtgtatagctttaaagcgccgaccagcgaagaatatgaacatgattttctgtggcgccattataaagcgctgccggaacgcggccgcattggcattcataaccgcagccattatgaaaacgtgctggtgtataaagtgcatccggaatatgtgctgaacgaaaacattccgggctatcaggatctgaaactgattgatgataaattttggaaaaaacgctatgaaagcattcgcagctttgaacagcatattaccgaaaacggcattaccattattaaaatttttctgaacgtgagcaaagaagaacagaaaagccgctttctggatcgcattaacgatccggcgaaaaactggaaatttagcagcagcgatattaccgaacgcggccgctgggatgaatatatgaaagcgtatgaaaccgcgattgaagaaaccagcaccgaacaggcgccgtggtatattattccggcggataaaaaatggcatgcgcgcctggcgattagccagattctggaagaacattttaaccgcctggatctgaaatttccggtgctggcggaagaagaagcgaaaaaactggatgaaattaaagaactgctgctgaaagaataa (SEQ ID NO: 9);

[0085]

[0086] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0087] Example 1: Enzyme Fermentation

[0088] The coding gene sequences for polypeptide ligase (HexPLigase), acetyltrans, acetyl-CoA synthase (PfAcCoASyn), ATP regenerase (PsPPK), and ammoniata (CpAmid) were synthesized separately. Then, the coding genes were subcloned into the pET28a plasmid through NdeI / XhoI restriction sites. The plasmid was then transformed into E. coli (BL21) cells for plate culture. Finally, the verified single clones were selected for liquid stepwise scale-up culture.

[0089] The steps for liquid scale-up culture are as follows: Transfer a single colony into 5 mL of LB medium containing 50 μM kanamycin (37) o C) Culture the cells. Once they reach the logarithmic growth phase, inoculate them into 250 mL of LB medium containing the same antibiotic, and then transfer them to a 5 L fermenter for further culture. When the cells reach the OD phase... 600 Add 0.5mM IPTG at 20°C 28°C o C-induced protein expression for 8 hours, followed by centrifugation (4000 rpm, 15 min) to collect 30-40 g of wet cells. Protein expression was then validated and enzyme activity was measured.

[0090] The steps for protein expression verification and enzyme activity assay are as follows: Take a small amount of cells and mix them evenly with Tris-HCl buffer (50mM, pH 8.0). Then, use the freeze-thaw method to lyse the cells. After high-speed centrifugation, take the supernatant and run it on an SDS-PAGE protein gel to confirm soluble protein expression. After confirming that the remaining cells are correct, mix them with the buffer (10g of wet cells mixed with about 200mL of the above buffer). Then, perform high-pressure cell lysis and high-speed centrifugation (16000rpm, 10min) to remove the cell wall. The resulting enzyme-containing supernatant (crude enzyme solution) can be used directly after measuring enzyme activity (enzyme activity assay results are shown in Table 1. The enzyme activity unit U is the amount of enzyme required to convert 1μmol of substrate in 1min at room temperature), or it can be further purified and immobilized for use (in solid enzyme reactions).

[0091] Table 1. Information on the properties of the enzymes used

[0092]

[0093] Example 2 Enzyme Immobilization

[0094] Ammonium sulfate was added incrementally to the collected crude enzyme solutions of HexPLigase, NkAcetylTrans, PfAcCoASyn, PsPPK, and CpAmid until enzyme precipitation (the amount of ammonium sulfate added was 40%–60% of the total enzyme-containing solutions, where % is w / v ammonium sulfate / buffer, where w is in g and v is in mL). The solutions were then collected by centrifugation (10,000 rpm, 15 min) and slowly dissolved in 25 mM pH 8.0 Tris buffer. Finally, the solutions were desalted using a G25 size exclusion column and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain pre-purified liquid enzymes HexPLigase, NkAcetylTrans, PfAcCoASyn, PsPPK, and CpAmid. In the immobilized mixed enzyme, the above-mentioned initially purified enzyme was immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of (1.5~2.5):(2.5~3.5):(2.5~3.5):(1.5~2.5):(1.5~2.5). The basic immobilization method was as follows: the mixed enzyme with a total enzyme activity of 10000U was dissolved in 2L of 50mM pH 8.0 potassium phosphate solution, followed by the addition of 60mM phenoxyacetic acid and 900g of LX-1000 EP epoxy resin to the buffer solution. After stirring at room temperature for 8 hours, the immobilized enzyme was filtered out and washed three times each with water and 25mM pH 8.0 phosphate buffer, and then dried at low temperature for later use. The HexPLigase, NkAcetylTrans, PfAcCoASyn, PsPPK, and CpAmid immobilized mixed enzymes exhibited 75%~89% of the activity of the corresponding liquid enzymes.

[0095] Example 3: Using tripeptides Glu-Glu-Met, Gln-Arg-Arg, and ATP as raw materials, hexapeptide Glu-Glu-Met-Gln-Arg-Arg was prepared by liquid enzymatic conversion with HexPLigase.

[0096] Add 13.4g Glu-Glu-Met (33mM), 13.8g Gln-Arg-Arg (30mM), 1.02g magnesium chloride hexahydrate (5mM), and 18.2g ATP (33mM) to 1L of 25mM pH 8.0 Tris-HCl solution, then adjust the pH back to 8.0. Then add 2000U of crude HexPLigase enzyme solution all at once. oAfter stirring the reaction gently for 2 hours, hydrochloric acid was added to terminate the reaction. The precipitate was removed by centrifugation. The solution was then adjusted to pH 7.0 and phosphate-containing impurities were removed using D201 anion exchange resin. The product-containing effluent was then purified and collected using D101 nonpolar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 21.1g of hexapeptide white solid (final yield 83%).

[0097] Example 4: Using tripeptides Glu-Glu-Met and Gln-Arg-Arg as raw materials, and employing an ATP regeneration system, hexapeptide Glu-Glu-Met-Gln-Arg-Arg was prepared by liquid enzyme (HexPLigase, PsPPK) conversion.

[0098] Similar to Example 3, but with the addition of ATP regenerase PsPPK to the reaction solution for cyclic regeneration.

[0099] Add 13.4g Glu-Glu-Met (33mM), 13.8g Gln-Arg-Arg (30mM), 1.02g magnesium chloride hexahydrate (5mM), 6.1g sodium hexametaphosphate (10mM), and 0.6g ATP (1mM) to 1L of 25mM pH 8.0 Tris-HCl solution, then adjust the pH back to 8.0. Then, add 2000U of crude HexPLigase enzyme solution and 2000U of crude PsPPK enzyme solution at once to initiate the reaction; 30 o After stirring the reaction gently for 4 hours, hydrochloric acid was added to terminate the reaction. The precipitate was removed by centrifugation, and the solution was then adjusted to pH 7.0. Phosphoric acid impurities were removed using D201 anion exchange resin. The product-containing effluent was then purified and collected using D101 nonpolar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 22.6g of hexapeptide white solid (final yield 89%).

[0100] Example 5: Acetylated hexapeptide Ac-Glu-Glu-Met-Gln-Arg-Arg was prepared using hexapeptide Glu-Glu-Met-Gln-Arg-Arg and acetyl-CoA as raw materials via liquid enzyme (NkAcetylTrans).

[0101] After adding 16.9 g of Glu-Glu-Met-Gln-Arg-Arg (20 mM), 17.8 g of acetyl-CoA (22 mM), and 1.02 g of magnesium chloride hexahydrate (5 mM) to 1 L of 25 mM pH 8.0 Tris-HCl solution, the pH of the solution was adjusted back to 8.0. Then, 2000 U of NkAcetylTrans crude enzyme solution was added all at once. The reaction solution was kept at 30°C.o C. Stir gently and maintain the pH of the reaction system between 7.0 and 9.0 using acid and base during the reaction. After 3 hours, the reaction is completed. Add acid to precipitate the enzyme in the reaction solution, and centrifuge quickly (10,000 rpm, 10 min) to remove the enzyme precipitate. Then, adjust the pH of the clarified solution back to 7.0 and use D201 anion exchange resin to remove impurities containing acetyl-CoA. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 15.5g of Ac-Glu-Glu-Met-Gln-Arg-Arg white solid (final yield 87%).

[0102] Example 6: Using the hexapeptide Glu-Glu-Met-Gln-Arg-Arg as a raw material, and in conjunction with an acetyl-CoA regeneration system, acetylated hexapeptide Ac-Glu-Met-Gln-Arg-Arg was prepared using liquid enzymes (NkAcetylTrans, PfAcCoASyn, PsPPK).

[0103] Add 35.6g Glu-Glu-Met-Gln-Arg-Arg (40mM), 0.8g acetyl-CoA (1mM), 2.0g magnesium chloride hexahydrate (10mM), 2.6g acetic acid (44mM), 0.6g ATP (1mM), and 8.6g sodium hexametaphosphate (14mM) to 1L of 25mM pH 8.0 Tris-HCl solution, then adjust the pH back to 8.0. Then, add 3000U NkAcetylTrans crude enzyme solution, 5000U PfAcCoASyn crude enzyme solution, and 2000U PsPPK crude enzyme solution at once to initiate the reaction. The reaction solution is kept at 30°C. o C. Stir gently and maintain the pH of the reaction system between 7.0 and 9.0 using acid and base during the reaction. After 4 hours, the reaction is completed. Add acid to precipitate the enzyme in the reaction solution, and centrifuge quickly (10,000 rpm, 10 min) to remove the enzyme precipitate. Then, adjust the pH of the clarified solution back to 7.0 and use D201 anion exchange resin to remove phosphate-containing impurities such as acetyl-CoA and adenosine. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 32.4 g of Ac-Glu-Glu-Met-Gln-Arg-Arg white solid (final yield 91%).

[0104] Example 7: Acetyl hexapeptide-8 was prepared using acetylated hexapeptide Ac-Glu-Glu-Met-Gln-Arg-Arg and ammonium chloride as raw materials, catalyzed by liquid enzyme (CpAmid).

[0105] Add 44.4g Ac-Glu-Glu-Met-Gln-Arg-Arg (50mM), 2.0g magnesium chloride hexahydrate (10mM), and 13.4g ammonium chloride (250mM) to 1L of 25mM pH 8.0 Tri-HCl solution to adjust the pH back to 8.0. Then, add 2000U of crude CpAmid enzyme solution at once to start the reaction; the reaction solution is kept at 30°C. o After stirring gently for 2 hours, the reaction was completed. Acid was added to precipitate the enzyme, and the precipitate was removed by centrifugation (10,000 rpm, 10 min). The pH of the clarified solution was then adjusted back to 7.0, and the crude Ac-Glu-Glu-Met-Gln-Arg-Arg product was collected by purification using D101 nonpolar resin. Finally, the product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 41.2 g of acetyl hexapeptide-8 white solid product (final yield 93%).

[0106] Example 8: Acetyl hexapeptide-8 was prepared from tripeptides Glu-Glu-Met, Gln-Arg-Arg, metahexaphosphate, and ammonium chloride using ATP, an acetyl-CoA regeneration system, and a liquid enzyme (HexPLigase, PsPPK, NkAcetylTrans, PfAcCoASyn, CpAmid) in a single conversion.

[0107] Add 8.9g Glu-Glu-Met (22mM), 9.2g Gln-Arg-Arg (20mM), 1.2g ATP (2mM), 0.8g acetyl-CoA (1mM), 4.1g magnesium chloride hexahydrate (20mM), 1.3g acetic acid (22mM), 9.2g sodium hexametaphosphate (15mM), and 5.4g ammonium chloride (100mM) to 1 L of 25mM pH 8.0 Tris-HCl solution, then adjust the pH of the solution back to 8.0. Then, add 2000U HexPLigase crude enzyme solution, 3000U NkAcetylTrans crude enzyme solution, 3000U PfAcCoASyn crude enzyme solution, 2000U PsPPK crude enzyme solution, and 2000U CpAmid crude enzyme solution to initiate the reaction. The reaction solution is kept at 30°C. oC. Stir gently and maintain the pH of the reaction system between 7.0 and 9.0 using acid and base during the reaction. After 5 hours, the reaction is completed. Add acid to precipitate the enzyme in the reaction solution, and centrifuge quickly (10,000 rpm, 10 min) to remove the enzyme precipitate. Then, adjust the pH of the clarified solution back to 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 15.1 g of acetyl hexapeptide-8 white solid (final yield 85%).

[0108] Example 9: Acetyl hexapeptide-8 was prepared by a single-stage conversion of an immobilized mixed enzyme using tripeptide Glu-Glu-Met, Gln-Arg-Arg, metahexaphosphate, and ammonium chloride as raw materials and an ATP- and acetyl-CoA regeneration system.

[0109] The reaction is similar to that in Example 8 above, but an immobilized enzyme is used, so it can be recycled multiple times.

[0110] After adding 8.9 g Glu-Glu-Met (22 mM), 9.2 g Gln-Arg-Arg (20 mM), 1.2 g ATP (2 mM), 0.8 g acetyl-CoA (1 mM), 4.1 g magnesium chloride hexahydrate (20 mM), 1.3 g acetic acid (22 mM), 9.2 g sodium hexametaphosphate (15 mM), and 5.4 g ammonium chloride (100 mM) to 1 L of 25 mM pH 8.0 Tris-HCl solution, the pH of the solution was adjusted back to 8.0. Finally, 10000 U of immobilized mixed enzyme was added all at once to initiate the reaction; the reaction was carried out at 35°C. o C. Stir gently and maintain the reaction pH at approximately 7.0–8.5 throughout the reaction. After 8 hours, the reaction is complete. The immobilized mixed enzyme is then collected by filtration (the mixed enzyme retains 88% of its initial activity after 6 uses). The filtrate is first treated with D201 anion exchange resin to remove phosphate-containing impurities such as acetyl-CoA and ADP. Then, the crude product is purified using D101 nonpolar resin. Finally, the product is desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 1:1, V:V) to obtain 15.8 g of white solid (final yield 89%).

[0111] Example 10: Acetyl hexapeptide-8 was prepared from tripeptides Glu-Glu-Met, Gln-Arg-Arg, metahexaphosphate, and ammonium chloride using ATP, an acetyl-CoA regeneration system, and liquid enzymes (WTHexPLigase, WTPsPPK, WTNkAcetylTrans, WTPfAcCoASyn, WTCpAmid) in a single conversion.

[0112] Similar to Example 8 above, each enzyme was replaced with the natural enzyme WT.

[0113] Add 8.9g Glu-Glu-Met (22mM), 9.2g Gln-Arg-Arg (20mM), 1.2g ATP (2mM), 0.8g acetyl-CoA (1mM), 4.1g magnesium chloride hexahydrate (20mM), 1.3g acetic acid (22mM), 9.2g sodium hexametaphosphate (15mM), and 5.4g ammonium chloride (100mM) to 1 L of 25mM pH 8.0 Tris-HCl solution, then adjust the pH back to 8.0. Then, add 5000U WTHexPLigase crude enzyme solution, 6000U WTNkAcetylTrans crude enzyme solution, 6000U WTPfAcCoASyn crude enzyme solution, 3000U WTPsPPK crude enzyme solution, and 5000U WTCpAmid crude enzyme solution to initiate the reaction. The reaction solution is kept at 30°C. o C. Stir gently and maintain the pH of the reaction system between 7.0 and 9.0 using acid and base during the reaction. After 12 hours, the reaction is completed. Add acid to precipitate the enzyme in the reaction solution, and centrifuge quickly (10,000 rpm, 10 min) to remove the enzyme precipitate. Then, adjust the pH of the clarified solution back to 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 1.3 g of acetyl hexapeptide-8 gray solid (final yield 7%).

[0114] 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 polypeptide ligase variant, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A combination enzyme, characterized in that, Including combination enzyme 1 and / or combination enzyme 2; The combined enzyme 1 comprises: the polypeptide ligase variant of claim 1, an acetylase or a variant thereof, and an ammonylase or a variant thereof; The combined enzyme 2 comprises: a polypeptide ligase variant of claim 1, an acetyltransferase or a variant thereof, an acetyl-CoA synthase or a variant thereof, an ATP regenerase or a variant thereof, and an ammonyltransferase or a variant thereof; The Uniprot ID of the acetyltransferase is A0A918XJD1; The Uniprot ID of the acetyl-CoA synthase is G0EH45; The Uniprot ID of the ATP regenerator is A0A1D7QNW6; The Uniprot ID of the amination enzyme is A0A1D7QNW6; The amino acid sequence of the variant of the acetyltransferase is shown in SEQ ID NO:2; The amino acid sequence of the variant of the acetyl-CoA synthase is shown in SEQ ID NO:3; The amino acid sequence of the variant of the ATP regenerating enzyme is shown in SEQ ID NO:4; The amino acid sequence of the variant of the ammoniata is shown in SEQ ID NO:

5.

3. A biomaterial, characterized in that, Includes at least one of the following: (I) to (V) I) Nucleic acids encoding the polypeptide ligase variant of claim 1 and / or the combinatorial enzyme of claim 2; II) Expression units containing nucleic acids as shown in I); III) Recombinant vectors containing nucleic acids as shown in I) or expression units as shown in II); IV) Integrating the genome into the nucleic acid shown in I) or the expression unit shown in II), or transfecting or transforming engineered cells with the recombinant vector shown in III); V) Cultures obtained by culturing engineered cells as shown in IV).

4. An immobilized enzyme, characterized in that, Including: immobilized enzyme 1 and / or immobilized enzyme 2; The immobilized enzyme 1 comprises: the polypeptide ligase variant and the vector as described in claim 1; The immobilized enzyme 2 comprises the combined enzyme and carrier as described in claim 2.

5. The immobilized enzyme according to claim 4, characterized in that, The carrier includes epoxy resin.

6. A composition, characterized in that, include: Composition A and / or Composition B; The composition A comprises: the combined enzyme and reactants as described in claim 2; The composition B comprises: immobilized enzyme 2 of the immobilized enzyme of claim 4 or 5 and reactants; The reactants include: reactant 1 and / or reactant 2; The reactant 1 includes: Glu-Glu-Met, Gln-Arg-Arg, magnesium chloride, ATP, acetyl-CoA and / or ammonium chloride; The reactant 2 includes: Glu-Glu-Met, Gln-Arg-Arg, ATP, acetyl-CoA, magnesium chloride, acetic acid, sodium hexametaphosphate, and / or ammonium chloride.

7. Use of at least one of the following shown in i) to iv) in the preparation of acetyl hexapeptide-8: i) The combined enzyme according to claim 2; ii) The biomaterial as described in claim 3; iii) The immobilized enzyme as described in claim 4 or 5; iv) The composition according to claim 6.

8. A method for preparing acetyl hexapeptide-8, characterized in that, To produce acetyl hexapeptide-8 using at least one of the following: A) to D) A) The combined enzyme as described in claim 2; B) The biomaterial as described in claim 3; C) Immobilized enzyme 2 in the immobilized enzyme of claim 4 or 5; D) The composition according to claim 6.

9. The preparation method according to claim 8, characterized in that, Includes the following steps: Acetyl hexapeptide-8 is synthesized using Glu-Glu-Met and Gln-Arg-Arg as substrates, with the participation of combinatorial enzyme 1 in the combinatorial enzyme of claim 2 and reactant 1 in the composition of claim 6; or Acetyl hexapeptide-8 was synthesized using Glu-Glu-Met and Gln-Arg-Arg as substrates, with the participation of combinatorial enzyme 2 in the combinatorial enzyme of claim 2 and reactant 2 in the composition of claim 6.

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