Enzyme composition and application thereof in preparation of acetyl hexapeptide-8

Abstract

The invention relates to the technical field of biology, in particular to an enzyme composition and application thereof in preparation of acetyl hexapeptide-8. The invention provides an enzymatic preparation process of acetyl hexapeptide-8, which is characterized in that after enzyme optimization, Glu-Glu-Met and Gln-Arg-Arg are taken as substrates, a hexapeptide skeleton is constructed under the catalysis of ATP-dependent polypeptide ligase, then acetylation modification is completed by N-terminal acetylase in the presence of acetyl CoA, and C-terminal amidation is realized by ammoniation enzyme. The preparation process can be implemented in sections or synthesized in one step, and continuous production can also be realized by adopting an immobilized enzyme reactor; the preparation process is used for synthesis of acetyl hexapeptide-8, has the core advantages of high economical efficiency, high process integration level, product purity of more than 99.5% and the like, and provides a new industrial normal form for green manufacturing of anti-wrinkle peptide raw materials.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an enzyme composition and application thereof in the preparation of acetyl hexapeptide-8. Background Art

[0002] The current preparation methods of acetyl hexapeptide-8 on the market are mainly concentrated in chemical methods, such as solid-phase synthesis, liquid-phase synthesis and segmented condensation strategies. For example, Lipotec uses solid-phase synthesis technology to gradually connect acetyl-glutamate-glutamate-methionine-glutamine-arginine-arginine sequences to form the target polypeptide chain and complete amino-terminal modification; Shandong Jipeptide uses a seven-step liquid-phase synthesis method to synthesize key intermediates such as Boc-Met-Gln-OH and Ac-Glu (OtBu) -Glu (OtBu) -OH in sequence, and finally obtains acetyl hexapeptide-8 by recrystallization purification; Hanpeptide Bio uses a segmented condensation strategy to synthesize three dipeptide monomers: Ac-Glu (O-tBu) -Glu (O-tBu) -OH, H-Met-Gln (Trt) -OH and H-Arg (Pbf) -Arg (Pbf) -NH2 through liquid phase, and then completes the directional connection synthesis of acetyl hexapeptide-8 through DCC / HOBt system.

[0003] However, the existing acetyl hexapeptide-8 synthesis method sequence cannot be accurately assembled, and there are peptide chain mismatches. In addition, the protective group removal step in traditional solid-phase synthesis produces many by-products, low purity, complex synthesis steps, and the yield needs to be further improved. Therefore, it is necessary to develop a new path for new grades of 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] The present invention provides a polypeptide ligase variant HexPLigase, whose amino acid sequence is shown in SEQ ID NO: 1.

[0006] The present invention provides a combination enzyme, which includes combination enzyme 1 and / or combination enzyme 2;

[0007] The enzyme combination 1 comprises: the polypeptide ligase variant HexPLigase of the present invention, the acetylase or its variant NkAcetylTrans, and the aminase or its variant CpAmid;

[0008] The enzyme combination 2 comprises: the polypeptide ligase variant HexPLigase of the present invention, acetylase or its variant NkAcetylTrans, acetyl-CoA synthetase or its variant PfAcCoASyn, ATP regenerator or its variant PsPPK, and aminase or its variant CpAmid;

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

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

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

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

[0013] Furthermore, in the combined enzyme of the present invention,

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

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

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

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

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

[0019] I), a nucleic acid encoding the polypeptide ligase variant HexPLigase of the present invention or the combined enzyme of the present invention;

[0020] II), an expression unit containing the nucleic acid as shown in I);

[0021] III), a recombinant vector containing the nucleic acid as shown in I) or the expression unit as shown in II);

[0022] IV), genomic integration of the nucleic acid as described in I) or the expression unit as described in II), or, transfection or transformation of a host cell containing the recombinant vector as described in III);

[0023] V) Cultivate the host cells obtained as described in IV).

[0024] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present 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 linear or circular in topology. The nucleic acid can be part of a vector (such as an expression or cloning vector) or a fragment. The nucleic acid can be obtained directly from a natural source, 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 the present invention, the nucleic acid may be optimized or unoptimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites 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 beginning with a promoter and ending with a terminator. The promoter and terminator may also be flanked or interposed with regulatory segments. These regulatory segments may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence, such as a promoter enhancer and poly(A) signal.

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

[0028] Furthermore, the vector backbone of the present invention may be derived from plants, animals, bacteria, fungi, phages, or viruses, but the present invention is not limited thereto. 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, pColdII, and the like. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, pAUR23, and the like.

[0029] Recombinant vector of the present invention refers to the nucleic acid vector of recombinant, is a kind of recombinant DNA molecule, it comprises desired coding sequence and the expression of operably connected coding gene in concrete host organism requisite suitable nucleotide sequence or element.In this manual, " plasmid " and " vector " can sometimes be interchangeable and general, because plasmid is the most commonly used vector form at present.Yet the present invention is intended to include such other forms of expression vector, it plays equivalent role, it is known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or is only potential genomic insert.In a specific embodiment, the nucleic acid encoding fusion protein provided by the invention can be constructed in various prokaryotic expression vectors.For example, its backbone vector can be pET28a.

[0030] The present invention provides a host cell for transformation or transfection of the recombinant vector. The host cell may be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, but the present invention is not limited thereto. In a specific embodiment of the present invention, the host cell is derived from bacteria; more specifically, Escherichia coli, more preferably E. coli (BL21) or E. coli DH5α.

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

[0032] The present invention provides an immobilized enzyme, which comprises at least one of the ligase variants of the present invention or the combined enzymes of the present invention and a carrier;

[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 enzyme of the present invention or the immobilized enzyme of 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 coenzyme A, magnesium chloride, acetic acid, sodium hexametaphosphate, and ammonium chloride.

[0039] The present 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 of the present invention;

[0041] ii), the combined enzyme of the present invention;

[0042] iii) the biomaterial of the present invention;

[0043] iv), the immobilized enzyme of the present invention;

[0044] v), the composition of the present invention.

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

[0046] a), the polypeptide ligase variant of the present invention;

[0047] b), the combined enzyme of the present invention;

[0048] c) the biomaterial of the present invention;

[0049] d), the immobilized enzyme of the present invention;

[0050] e) The composition of the present invention.

[0051] The present invention provides a method for preparing acetyl hexapeptide-8, characterized in that acetyl hexapeptide-8 is produced using at least one of the following A) to E):

[0052] A), the polypeptide ligase variant of the present invention;

[0053] B), the combined enzyme of the present invention;

[0054] C), the biomaterial of the present invention;

[0055] D), the immobilized enzyme of the present invention;

[0056] E), the composition of the present invention.

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

[0058] Using Glu-Glu-Met and Gln-Arg-Arg as substrates, synthesizing acetyl hexapeptide-8 with the participation of the combined enzyme 1 in the combined enzyme of the present invention and the reactant 1 in the composition of the present invention; or

[0059] Acetyl hexapeptide-8 is synthesized with Glu-Glu-Met and Gln-Arg-Arg as substrates in the presence of the combined enzyme 2 in the combined enzyme of the present invention and the reactant 2 in the composition of the present invention.

[0060] In the present invention, the method for preparing acetyl hexapeptide-8 can synthesize acetyl hexapeptide-8 from the substrates Glu-Glu-Met and Gln-Arg-Arg in one step with the participation of the above-mentioned combined enzyme and reactants, or synthesize acetyl hexapeptide-8 through a multi-step reaction, which is not limited by the present invention.

[0061] Specifically, when the combined enzyme can be directly mixed, it can also be an immobilized enzyme formed by immobilization; in the immobilized enzyme system, the polypeptide ligase (HexPLigase), acetylase (NkAcetylTrans), acetyl-CoA synthetase (PfAcCoASyn), ATP regenerating enzyme (PsPPK) and aminase (CpAmid) are mixed and fixed according to the unit activity ratio of (1.5-2.5): (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5); the specific unit activity ratio of 2:3:3:2:2 is the best effect of mixing and fixing;

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

[0063] The experimental results showed that all of the above methods can achieve the synthesis of acetyl hexapeptide-8 with high purity and low byproducts; however, the final yield was significantly improved when using the variant enzymes compared to the wild-type enzyme. The one-step method for synthesizing acetyl hexapeptide-8 has the advantages of high purity and high yield while simplifying the steps. Combined with the immobilized enzyme, the enzyme can be used multiple times, further reducing production costs.

[0064] This invention provides an efficient and precise enzymatic preparation process for acetyl hexapeptide-8. Using two tripeptides, Glu-Glu-Met and Gln-Arg-Arg, as raw materials, the hexapeptide backbone is constructed under the catalysis of an ATP-dependent peptide ligase (HexPLigase). Subsequently, acetylation is achieved by an N-terminal acetyltransferase (NkAcetylTrans) in the presence of acetyl-CoA, and the C-terminus is amidated by an aminotransferase (CpAmid). This process can be implemented in stages or continuously using an immobilized enzyme reactor. To address the high costs of ATP and acetyl-CoA, an innovative ATP regeneration system driven by ATP regeneration enzyme (PsPPK) and an acetyl-CoA synthetase (PfAcCoASyn)-coupled acetyl-CoA recycling system were developed, reducing cofactor consumption by 85%. The HexPLigase, modified through directed evolution, has increased catalytic efficiency by over 20-fold and reduced byproduct generation to below 0.5%. With an optimized activity ratio (2:3:2), the three-enzyme system can achieve a single-pot conversion rate exceeding 80%. This technology combines core advantages such as high atomic economy, strong process integration, and product purity >99.5%, providing a new industrial paradigm for the green manufacturing of anti-wrinkle peptide raw materials.

[0065] The multi-enzyme complex system preparation method used in this patent is different from the common chemical synthesis method on the market today. For the first time, Glu-Glu-Met, Gln-Arg-Arg, magnesium chloride hexahydrate, acetic acid, sodium hexametaphosphate and other cheap bulk products are used as initial raw materials, and an independently developed multi-enzyme system is used for high-yield, high-quality splicing and modification to obtain acetyl hexapeptide-8, providing a new idea for the preparation of acetyl hexapeptide-8 by this multi-enzyme complex system.

[0066] The present invention provides an enzymatic preparation process for acetyl hexapeptide-8. Following 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 peptide ligase. Subsequently, acetylation modification is completed by an N-terminal acetylase in the presence of acetyl CoA, and C-terminal amidation is achieved by an aminonase. The process can be implemented in stages or in a single step, and continuous production can also be achieved using an immobilized enzyme reactor. The process, used for the synthesis of acetyl hexapeptide-8, combines core advantages such as high cost-effectiveness, strong process integration, and product purity exceeding 99.5%, providing a new industrial paradigm for the green manufacturing of anti-wrinkle peptide raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The figure shows the SDS-PAGE gel detection after enzyme purification, where M is a protein standard marker; 1 is HexPLigase; 2 is NkAcetylTrans; 3 is PfAcCoASyn; 4 is PsPPK; 5 is CpAmid;

[0068] Figure 2 The HPLC chromatogram of acetyl hexapeptide-8 after purification is shown. The detection conditions are: 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 o C, 215 nm detection;

[0069] Figure 3 Figure 4 shows the mass spectrum of acetyl hexapeptide-8, Agilent Ultivo LC / TQ. DETAILED DESCRIPTION

[0070] The present invention provides an enzyme composition and its use in the preparation of acetyl hexapeptide-8. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0071] Polypeptide ligase (HexPLigase): Derived from Saccharothrix australiensis (Uniprot ID: A0A495VZ10), this natural enzyme (WTHexPLigase) has low activity towards the substrate. After system modification (HexPLigase), its activity and expression level have been improved. The specific mutation sites are: W22T, G122M, H123F, L131V, S132N, Q133A, K231R, D248W, P284S, M305K, R306G and E325R.

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

[0073] Acetyl-CoA synthetase (PfAcCoASyn): Derived from Pyrolobus fumarii (Uniprot ID: G0EH45), this natural enzyme (WTPfCoASyn) has a high ability to regenerate acetyl-CoA, but its expression level is relatively poor; through the modification of the enzyme structure (PfAcCoASyn), its activity and expression have been significantly improved, and its mutation sites are: H36T, Y123I, R208P, F209Q, R286N and N428S.

[0074] ATP regeneration enzyme (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 very ideal. After site-directed mutagenesis at several sites (PsPPK), its stability has been greatly improved. The specific mutation sites are: M67L, Q89E, C133Y and S174T.

[0075] Aminase (CpAmid): Derived from the sea ice psychrophile bacterium Colwellia sp. (Uniprot ID: A0A1D8RSI1), this natural enzyme (WTCpAmid) has low substrate activity and low expression levels. Through systematic experimental optimization and modification, a mutant enzyme (CpAmid) with improvements 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 the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0087] Example 1 Enzyme fermentation

[0088] The coding gene sequences corresponding to polypeptide ligase (HexPLigase), acetylase (NkAcetylTrans), acetyl-CoA synthetase (PfAcCoASyn), ATP regenerator (PsPPK), and aminase (CpAmid) were synthesized respectively, and then subcloned into the pET28a plasmid through the NdeI / XhoI restriction sites. The plasmid was transferred into E. coli (BL21) cells for plate culture, and finally the correct single clone was selected for liquid step-by-step amplification culture.

[0089] The steps of liquid amplification culture are as follows: transfer a single colony into 5 mL of LB culture medium containing 50 μM kanamycin (37 o C) Cultivate the cells. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotics, and then transfer them into a 5 L culture fermenter for cultivation. When the cell OD 600 Add 0.5mM IPTG at 20 ℃ for 28 o Protein expression was induced by C for 8 h, and then the wet cells were collected by centrifugation (4000 rpm, 15 min) at 30-40 g. Protein expression was then verified and enzyme activity was determined.

[0090] Protein expression verification and enzyme activity assays were performed as follows: a small amount of cells was mixed evenly with Tris-HCl buffer (50 mM, pH 8.0), then disrupted by freeze-thaw. After high-speed centrifugation, the supernatant was run on an SDS-PAGE gel to confirm soluble protein expression. The remaining cells, confirmed to be correct, were mixed with buffer (10 g of wet cells mixed with approximately 200 mL of the aforementioned buffer), then disrupted by high-pressure pressure and centrifuged at high speed (16,000 rpm for 10 min) to remove the cell walls. The resulting enzyme-containing supernatant (crude enzyme solution) was assayed for enzyme activity and used directly (enzyme activity assay results are shown in Table 1; the enzyme activity unit (U) is the amount of enzyme required to convert 1 μmol of substrate in 1 min at room temperature) or further purified and immobilized for use (for solid enzyme reactions).

[0091] Table 1. Enzyme properties used

[0092]

[0093] Example 2 Enzyme immobilization

[0094] Ammonium sulfate solid was added incrementally to the crude enzyme solutions of polypeptide ligase (HexPLigase), acetyltransferase (NkAcetylTrans), acetyl-CoA synthetase (PfAcCoASyn), ATP regenerating enzyme (PsPPK), and aminase (CpAmid) until the enzymes precipitated (the amount of ammonium sulfate solid added was 40%–60% of the total enzyme-containing supernatant, where % represents w / v ammonium sulfate / buffer, where w is g and v is mL). The solution was then collected by centrifugation (10,000 rpm, 15 min) and slowly dissolved in 25 mM Tris buffer (pH 8.0). Finally, the solution was desalted on a G25 size exclusion chromatography column and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the pre-purified liquid enzymes HexPLigase, NkAcetylTrans, PfAcCoASyn, PsPPK, and CpAmid. In the immobilized enzyme mix, the pre-purified enzymes were immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) in a 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 enzyme mix, obtained by mixing the above activity units in the above ratios, yielding a total enzyme activity of 10,000 U, was dissolved in 2 L of 50 mM potassium phosphate solution, pH 8.0. 60 mM phenoxyacetic acid and 900 g of LX-1000EP epoxy resin were then added to the buffer. After stirring at room temperature for 8 hours, the immobilized enzymes were filtered and washed three times with water and three times with 25 mM phosphate buffer, pH 8.0, before being dried at low temperature for later use. The immobilized enzyme mixes for HexPLigase, NkAcetylTrans, PfAcCoASyn, PsPPK, and CpAmid exhibited 75%-89% of the activity of the corresponding liquid enzymes.

[0095] Example 3 Using the tripeptide Glu-Glu-Met, Gln-Arg-Arg and ATP as raw materials, HexPLigase liquid enzyme was used to convert the hexapeptide Glu-Glu-Met-Gln-Arg-Arg

[0096] After adding 13.4 g Glu-Glu-Met (33 mM), 13.8 g Gln-Arg-Arg (30 mM), 1.02 g magnesium chloride hexahydrate (5 mM) and 18.2 g ATP (33 mM) to 1 L 25 mM pH 8.0 Tris-HCl solution, the pH value of the solution was adjusted back to 8.0, and then 2000 U HexPLigase crude enzyme solution was added at once for 30 min. oC was stirred gently for 2 hours, and then hydrochloric acid was added to terminate the reaction. The protein was precipitated and centrifuged to remove the protein. The solution was then adjusted to pH 7.0 and the phosphoric acid-containing impurities were removed using D201 anion exchange resin. The effluent containing the product was then purified and collected using D101 non-polar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: H2O = 1:1, V:V) to obtain 21.1 g of hexapeptide as a white solid (final yield 83%).

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

[0098] Similar to Example 3, but ATP regeneration enzyme PsPPK was added to the reaction solution to recycle it.

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

[0100] Example 5 Using hexapeptide Glu-Glu-Met-Gln-Arg-Arg and acetyl-CoA as raw materials, liquid enzyme (NkAcetylTrans) was used to prepare acetylated hexapeptide Ac-Glu-Glu-Met-Gln-Arg-Arg

[0101] After adding 16.9 g Glu-Glu-Met-Gln-Arg-Arg (20 mM), 17.8 g acetyl-CoA (22 mM) and 1.02 g magnesium chloride hexahydrate (5 mM) to 1 L 25 mM pH 8.0 Tris-HCl solution, the pH value of the solution was adjusted back to 8.0. Then, 2000 U NkAcetylTrans crude enzyme solution was added at once. The reaction solution was incubated at 30o C was gently stirred, and the pH of the reaction system was maintained between 7.0 and 9.0 with acid and base during the reaction. The reaction was completed after 3 h, and acid was added to precipitate the enzyme in the reaction solution. The enzyme precipitate was removed by rapid centrifugation (10,000 rpm, 10 min). The pH of the clarified solution was then adjusted back to 7.0, and impurities containing acetyl-CoA were removed using D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg was then purified and collected using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=1:1, V:V) to obtain 15.5 g of Ac-Glu-Glu-Met-Gln-Arg-Arg as a white solid (final yield 87%).

[0102] Example 6 Using the hexapeptide Glu-Glu-Met-Gln-Arg-Arg as raw material, the acetyl-CoA regeneration system and liquid enzyme (NkAcetylTrans, PfAcCoASyn, PsPPK) were used to prepare the acetylated hexapeptide Ac-Glu-Glu-Met-Gln-Arg-Arg

[0103] To 1 L of 25 mM pH 8.0 Tris-HCl solution, 35.6 g Glu-Glu-Met-Gln-Arg-Arg (40 mM), 0.8 g acetyl-CoA (1 mM), 2.0 g magnesium chloride hexahydrate (10 mM), 2.6 g acetic acid (44 mM), 0.6 g ATP (1 mM), and 8.6 g sodium hexametaphosphate (14 mM) were added to adjust the pH of the solution back to 8.0. 3000 U of NkAcetylTrans crude enzyme solution, 5000 U of PfAcCoASyn crude enzyme solution, and 2000 U of PsPPK crude enzyme solution were then added all at once to initiate the reaction; the reaction solution was incubated at 30 o C was stirred gently, and the pH of the reaction system was maintained between 7.0 and 9.0 with acid and base during the reaction. The reaction was completed after 4 hours, and acid was added to precipitate the enzyme in the reaction solution. The enzyme precipitate was removed by rapid centrifugation (10,000 rpm, 10 min). The pH value of the clarified solution was then adjusted back to 7.0, and acetyl-CoA, adenosine and other phosphate-containing impurities were removed using D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg was then purified and collected using D101 non-polar resin. Finally, the crude product was 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 as a white solid (final yield 91%).

[0104] Example 7 Preparation of acetyl hexapeptide-8 using acetylated hexapeptide Ac-Glu-Glu-Met-Gln-Arg-Arg and ammonium chloride as raw materials and catalysis by liquid enzyme (CpAmid)

[0105] After adding 44.4 g Ac-Glu-Glu-Met-Gln-Arg-Arg (50 mM), 2.0 g magnesium chloride hexahydrate (10 mM), and 13.4 g ammonium chloride (250 mM) to 1 L 25 mM pH 8.0 Tri-HCl solution, the pH value of the solution was adjusted back to 8.0. Then, 2000 U CpAmid crude enzyme solution was added at once to start the reaction. The reaction solution was heated at 30 o The reaction was terminated after gentle stirring for 2 hours at C. Acid was added to precipitate the reaction enzyme, and the enzyme precipitate was removed by centrifugation (10,000 rpm, 10 minutes). The pH value of the clarified liquid was then adjusted back to 7.0, and the crude Ac-Glu-Glu-Met-Gln-Arg-Arg product was purified and collected using D101 non-polar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: H2O = 1:1, V:V) to obtain 41.2 g of acetyl hexadecapeptide-8 as a white solid product (final yield 93%).

[0106] Example 8: Acetyl hexapeptide-8 was prepared by one-step conversion using tripeptide Glu-Glu-Met, Gln-Arg-Arg, hexaphosphate, and ammonium chloride as raw materials using ATP, acetyl-CoA regeneration system, and liquid enzymes (HexPLigase, PsPPK, NkAcetylTrans, PfAcCoASyn, and CpAmid).

[0107] To 1 L of 25 mM pH 8.0 Tris-HCl solution, 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) were added to adjust the pH of the solution back to 8.0. Then, 2000 U HexPLigase crude enzyme solution, 3000 U NkAcetylTrans crude enzyme solution, 3000 U PfAcCoASyn crude enzyme solution, 2000 U PsPPK crude enzyme solution, and 2000 U CpAmid crude enzyme solution were added all at once to initiate the reaction. The reaction solution was incubated at 30 oC was stirred gently, and the pH of the reaction system was maintained between 7.0 and 9.0 with acid and alkali during the reaction. The reaction was completed after 5 hours, and acid was added to precipitate the enzyme in the reaction solution. The enzyme precipitate was removed by rapid centrifugation (10000 rpm, 10 min), and then the pH value of the clarified liquid was adjusted back to 7.0 and the phosphate-containing impurities were removed by D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 was then purified and collected by D101 non-polar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: H2O=1:1, V:V) to obtain 15.1 g of acetyl hexadecapeptide-8 as a white solid (final yield 85%).

[0108] Example 9: Using tripeptide Glu-Glu-Met, Gln-Arg-Arg, hexaphosphate, and ammonium chloride as raw materials, ATP, acetyl coenzyme A regeneration system, and immobilized mixed enzymes for one-time conversion to prepare acetyl hexapeptide-8

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

[0110] 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) were added to 1 L of 25 mM pH 8.0 Tris-HCl solution, and the pH value of the solution was adjusted back to 8.0. Finally, 10,000 U of immobilized enzyme mixture was added at once to start the reaction; the reaction was continued at 35 o C was gently stirred, and the reaction pH was maintained at approximately 7.0-8.5 throughout the reaction. The reaction was complete after 8 h, and the immobilized enzyme mixture was then collected by filtration (the enzyme mixture retained 88% of its initial activity after 6 uses). The filtrate was first subjected to D201 anion exchange resin to remove phosphate-containing impurities such as acetyl-CoA and ADP, and then purified using D101 non-polar resin to collect the crude product. Finally, the product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=1:1, V:V) to obtain 15.8 g of a white solid (final yield 89%).

[0111] Example 10: Acetyl hexapeptide-8 was prepared by one-step conversion using tripeptide Glu-Glu-Met, Gln-Arg-Arg, hexaphosphate, and ammonium chloride as raw materials using ATP, acetyl-CoA regeneration system, and liquid enzymes (WTHexPLigase, WTPsPPK, WTNkAcetylTrans, WTPfAcCoASyn, and WTCpAmid).

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

[0113] To 1 L of 25 mM pH 8.0 Tris-HCl solution, 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) were added to adjust the pH of the solution back to 8.0. Then, 5000 U of WTHexPLigase crude enzyme solution, 6000 U of WTNkAcetylTrans crude enzyme solution, 6000 U of WTPfAcCoASyn crude enzyme solution, 3000 U of WTPsPPK crude enzyme solution, and 5000 U of WTCpAmid crude enzyme solution were added all at once to initiate the reaction. The reaction solution was incubated at 30 o C with gentle stirring, and the pH of the reaction system was maintained between 7.0 and 9.0 with acid and base during the reaction; the reaction was completed after 12 hours, and acid was added to precipitate the enzyme in the reaction solution. The enzyme precipitate was removed by rapid centrifugation (10,000 rpm, 10 min), and then the pH value of the clarified liquid was adjusted back to 7.0 and the phosphate-containing impurities were removed using D201 anion exchange resin. The effluent containing Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 was then purified and collected using D101 non-polar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: H2O=1:1, V:V) to obtain 1.3 g of acetyl hexadecapeptide-8 as a gray solid (final yield 7%).

[0114] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as 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 combined enzyme, characterized in that including combined enzyme 1 and / or combined enzyme 2; The combined enzyme 1 comprises: the polypeptide ligase variant, acetylase or a variant thereof, and aminase or a variant thereof according to claim 1; The enzyme combination 2 comprises: the polypeptide ligase variant according to claim 1, an acetylase or a variant thereof, an acetyl-CoA synthetase or a variant thereof, an ATP regenerator or a variant thereof, and an aminase or a variant thereof; The Uniprot ID of the acetylase is A0A918XJD1; The Uniprot ID of the acetyl-CoA synthetase is G0EH45; The Uniprot ID of the ATP regenerating enzyme is A0A1D7QNW6; The Uniprot ID of the ammonia enzyme is A0A1D7QNW6.

3. The combined enzyme according to claim 2, characterized in that The amino acid sequence of the variant of the acetylase is shown in SEQ ID NO: 2; The amino acid sequence of the variant of acetyl-CoA synthetase 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 aminomethane variant is shown in SEQ ID NO:

5.

4. Biomaterial, characterized in that Including at least one of the following I) to V): 1), a nucleic acid encoding the polypeptide ligase variant according to claim 1 and / or the combined enzyme according to claim 2 or 3; II), an expression unit containing the nucleic acid as shown in I); III), a recombinant vector containing the nucleic acid as shown in I) or the expression unit as shown in II); IV), genomic integration of the nucleic acid as described in I) or the expression unit as described in II), or, transfection or transformation of a host cell containing the recombinant vector as described in III); V) Cultivate the host cells obtained as described in IV).

5. An immobilized enzyme, characterized in that The method comprises at least one of the ligase variant according to claim 1 and / or the combined enzyme according to claim 2 or 3 and a carrier.

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

7. A composition characterized in that Comprising at least one of the combined enzyme according to claim 2 or 3 and / or the immobilized enzyme according to claim 5 or 6 and a reactant; The reactant 1 includes reactant 1 and reactant 2; The reactant 1 includes: Glu-Glu-Met, Gln-Arg-Arg, magnesium chloride, ATP, acetyl-CoA, CpAmid and / or ammonium chloride; The reactant 2 includes: Glu-Glu-Met, Gln-Arg-Arg, ATP, acetyl coenzyme A, magnesium chloride, acetic acid, sodium hexametaphosphate and / or ammonium chloride.

8. Use of at least one of the following i) to v) in the preparation of acetyl hexapeptide-8: i), the polypeptide ligase variant according to claim 1; ii), the combined enzyme according to claim 2 or 3; iii) the biomaterial according to claim 4; iv), the immobilized enzyme according to claim 5 or 6; v) The composition according to claim 7.

9. A product containing acetyl hexapeptide-8, characterized in that: The production raw materials include at least one of the following a) to e): a), the polypeptide ligase variant according to claim 1; b) The combined enzyme according to claim 2 or 3; c) The biomaterial according to claim 4; d) The immobilized enzyme according to claim 5 or 6; e) The composition according to claim 7.

10. A method for preparing acetyl hexapeptide-8, characterized in that: To produce acetyl hexapeptide-8 using at least one of the following A) to E) A), the polypeptide ligase variant according to claim 1; B), the combined enzyme according to claim 2 or 3; C) The biomaterial according to claim 4; D), the immobilized enzyme according to claim 5 or 6; E) The composition according to claim 7.

11. The preparation method according to claim 10, characterized in that: The steps include: Using Glu-Glu-Met and Gln-Arg-Arg as substrates, synthesizing acetyl hexapeptide-8 in the presence of the combined enzyme 1 in the combined enzyme according to claim 2 or 3 and the reactant 1 in the composition according to claim 9; or Acetyl hexapeptide-8 is synthesized with Glu-Glu-Met and Gln-Arg-Arg as substrates with the participation of the combined enzyme 2 in the combined enzyme according to claim 2 or 3 and the reactant 2 in the composition according to claim 9.

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