Enzyme mutants and their application in the preparation of conopodin

Abstract

This application relates to the field of biochemistry, and particularly to enzyme mutants and their application in the preparation of conopod peptides. This invention employs a strategy combining green chemistry and enzymatic methods, using seven tripeptide fragments as raw materials to prepare the conopod peptide backbone via liquid-phase synthesis. Then, a multi-redirecting evolutionary enzyme synergistic catalytic system is utilized to achieve precise enzymatic modification and synthesis. Based on the efficient linkage characteristics of the liquid-phase synthesis system and the high substrate specificity of the enzymatic system, this invention effectively avoids the complex protecting group operations in traditional solid-phase synthesis, significantly reduces byproduct generation, and simultaneously achieves site-specific modification and precise assembly of the conopod peptide backbone, providing an efficient and green new pathway for the continuous production of cosmetic-grade high-purity conopod peptides.

Description

Technical Field

[0001] This application relates to the field of biochemistry, and in particular to the preparation of conopodeptides using a chemical enzyme fusion process. Background Technology

[0002] Conus peptide is a neuromodulatory peptide extracted from the venom of marine cone snails, possessing both highly effective anti-wrinkle and neuroprotective properties. Its mechanism of action involves precisely blocking nerve signal transmission, inhibiting excessive muscle contraction, thereby reducing the formation of dynamic wrinkles; simultaneously, it activates skin repair pathways, promotes the regeneration of collagen and elastin, and enhances the skin's structural support. In cosmetic applications, conus peptide effectively relaxes dynamic facial muscles by regulating calcium ion channels at the neuromuscular junction, quickly smoothing deep expression lines such as frown lines and glabellar lines; its anti-wrinkle effect can last for more than 48 hours, offering both immediate smoothing and long-lasting firming benefits; furthermore, this ingredient can alleviate skin sensitivity and redness by inhibiting the release of inflammatory factors, improving the skin barrier function. Based on its unique synergistic effect of neuromodulation and skin regeneration, conus peptide has significant application potential in anti-aging skincare products, medical aesthetic anti-wrinkle care, and the repair of neurodermatitis, especially suitable for high-end skincare needs requiring both soothing and firming effects.

[0003] Currently, the main methods for preparing cone snail peptides on the market include chemical synthesis, biosynthesis based on recombinant DNA technology, and direct extraction from natural cone snails. For example, using solid-phase synthesis with Rink resin as a carrier, amino acids modified with Fmoc protecting groups are gradually coupled to form linear cone snail toxin precursors. After deprotection by cleavage with a mixed reagent such as phenol / dimercaptoacetic acid / TFA, disulfide bonds are formed through correct folding, ultimately yielding bioactive cone snail toxin peptides (CN1120174 C-Preparation method of marine cone snail analgesic peptides). Shenzhen BGI extracts crude venom from the venom tubes of barrel-shaped cone snails, extracts it with a solution containing 30% acetonitrile and 0.1% trifluoroacetic acid, enriches the target peptides through reductive alkylation and solid-phase extraction column chromatography, and then purifies and identifies its amino acid sequence using high-performance liquid chromatography, finally obtaining the active cone snail toxin κ-CPTx-btl01 (CN 107074909 B-Cone snail toxin peptide κ-CPTx-btl01, its preparation method and application). Alternatively, a recombinant vector containing a GST tag and an enterokinase recognition sequence can be constructed using gene recombination technology. The fusion protein is expressed in Escherichia coli, and high-purity μ-conotoxin is obtained by purification by GST affinity chromatography, enterokinase cleavage, and ion exchange chromatography (CN 116355932 B-Recombinant Vector and Method for Preparing μ-Conotoxin).

[0004] However, solid-phase synthesis requires repeated protection group operations, resulting in cumbersome processes, numerous byproducts, and reliance on toxic solvents, causing serious environmental pollution; liquid-phase stepwise synthesis is limited by multi-step intermediate synthesis and purification, resulting in low efficiency and high cost; enzymatic ligation methods have limited substrate compatibility and are prone to producing sequence mismatch byproducts; while recombinant protein technology faces bottlenecks such as low expression levels and complex purification processes. Summary of the Invention

[0005] In view of this, this application provides a chemical-enzymatic fusion process for preparing conopeptides. Employing a strategy combining green chemistry and enzymatic methods, it uses seven tripeptide fragments, including Fmoc-Arg(Pbf)-Cys(Trt)-Cys(Trt)-OH and Fmoc-Asp(OtBu)-His(Trt)-Ala-OH, as raw materials. The conopeptide backbone is prepared via liquid-phase synthesis, and then a multi-redirecting evolutionary enzyme synergistic catalytic system is used to achieve precise enzymatic modification and synthesis. Based on the efficient linkage characteristics of the liquid-phase synthesis system and the high substrate specificity of the enzymatic system, this process effectively avoids the complex protecting group operations in traditional solid-phase synthesis, significantly reduces byproduct generation, and simultaneously achieves site-specific modification and precise assembly of the conopeptide backbone. This provides an efficient and green new pathway for the continuous production of cosmetic-grade high-purity conopeptides.

[0006] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0007] This application provides any one of the following: a pyroglutamate ligase with the amino acid sequence SEQ ID NO: 1, a thiol oxidase with the amino acid sequence SEQ ID NO: 2, an ammonia-transferase with the amino acid sequence SEQ ID NO: 3, a lactate dehydrogenase with the amino acid sequence SEQ ID NO: 4, and an ATP regenerator with the amino acid sequence SEQ ID NO: 5.

[0008] This application also provides an enzyme combination comprising at least two of the following: a pyroglutamate ligase with the amino acid sequence SEQ ID NO: 1, a thiol oxidase with the amino acid sequence SEQ ID NO: 2, an ammonia-transferase with the amino acid sequence SEQ ID NO: 3, a lactate dehydrogenase with the amino acid sequence SEQ ID NO: 4, and an ATP regenerator with the amino acid sequence SEQ ID NO: 5.

[0009] This application also provides immobilized enzymes comprising the above-described enzyme combination.

[0010] This application also provides a method for preparing the above-mentioned immobilized enzyme, including:

[0011] The pyroglutamate ligase with amino acid sequence SEQ ID NO: 1, the thiol oxidase with amino acid sequence SEQ ID NO: 2, the ammonia-transferase with amino acid sequence SEQ ID NO: 3, the lactate dehydrogenase with amino acid sequence SEQ ID NO: 4, and the ATP regenerator with amino acid sequence SEQ ID NO: 5 were mixed to obtain a mixed enzyme.

[0012] The mixed enzyme was dissolved in potassium phosphate solution, then mixed with phenoxyacetic acid and epoxy resin, stirred, and filtered to obtain immobilized enzyme.

[0013] In some specific embodiments of this application, the pyroglutamate ligase, thiol oxidase, lactate dehydrogenase, ATP regenerating enzyme, and amination enzyme in the above-mentioned immobilized enzyme preparation method are mixed in an enzyme activity unit ratio of 2:(1~3):(1~3):(1~3):(1~3) (which can be 2:2:2:2:2.8, 2:2:2:2:2.9, 2:2:2:2:3.1 or 2:2:2:2:3.2), and the enzyme activity unit of the mixed enzyme is 5000~15000 U (which can be 7000 U, 9000 U, 11000 U or 13000 U);

[0014] The potassium phosphate solution has a concentration of 40-60 mM (which can be 45 mM, 48 mM, 62 mM or 65 mM), a pH of 7.5-8.5 (which can be 7.8, 7.9, 8.1 or 8.2), and a volume of 1-3 L (which can be 1.8 L, 1.9 L, 2.1 L or 2.2 L).

[0015] The final concentration of the phenoxyacetic acid is 40-60 mM (which can be 45 mM, 48 mM, 62 mM or 65 mM).

[0016] The epoxy resin is LX-1000 EP epoxy resin, with a weight of 800~1000 grams (it can be 880 grams, 890 grams, 910 grams or 920 grams).

[0017] The stirring time is 6 to 10 hours (it can be 7 hours, 7.5 hours, 8.5 hours or 9 hours).

[0018] This application also provides the application of the above-mentioned enzyme combination, the above-mentioned immobilized enzyme, or the immobilized enzyme prepared by the above-mentioned immobilized enzyme preparation method in the preparation of conotoxin.

[0019] This application also provides a method for preparing conospirin, including:

[0020] Buffer, conopeptide backbone, pyruvate and NAD +Mix, adjust the pH to 7.0~9.0 (it can be 7.5, 7.9, 8.1 or 8.5), and then mix with thiol oxidase with amino acid sequence SEQ ID NO: 2 and lactate dehydrogenase with amino acid sequence SEQ ID NO: 4 to obtain mixture 1. Stir, mix with acid, separate and purify to obtain oxidized conospirin backbone;

[0021] Mix buffer, oxidized conospirin backbone, pyroglutamic acid, sodium hexametaphosphate, magnesium chloride, and ATP, and adjust the pH to 7.0–9.0 (it can be 7.5, 7.9, 8.0, 8.1, or 8.5). Then mix with pyroglutamic acid ligase with amino acid sequence SEQ ID NO: 1 and ATP regenerator with amino acid sequence SEQ ID NO: 5 to obtain mixture 2. Stir and maintain the pH at 7.5–9.0 (it can be 7.8, 7.9, 8.0, 8.1, or 8.5). Mix with acid, separate and purify to obtain pyroglutaminated conospirin backbone.

[0022] Mix the buffer solution, the pyroglutamic conospirin backbone, and concentrated ammonia, adjust the pH to 7.0-9.0 (it can be 7.5, 7.9, 8.0, 8.1, or 8.5), and then mix with an aminotransferase with the amino acid sequence SEQ ID NO: 3 to obtain mixture 3. Stir, mix with acid, separate and purify to obtain conospirin.

[0023] In some specific embodiments of this application, the buffer solution in the above-mentioned method for preparing conospirin is 0.8~1.2 L (which can be 0.9 L, 0.95 L, 1.05 L or 1.1 L), 20~30 mM (which can be 23 mM, 24 mM, 26 mM or 27 mM), pH 7.5~8.5 (which can be 7.8, 7.9, 8.1 or 8.2), and tris(hydroxymethyl)aminomethane hydrochloric acid;

[0024] The content of the conotoxin backbone in the mixture 1 is 8~12 mM (which can be 9 mM, 9.5 mM, 10.5 mM or 11 mM).

[0025] The pyruvate content in the mixture 1 is 26.4~39.6 mM (which can be 28 mM, 32 mM, 35 mM or 37 mM).

[0026] The NAD + The content in the mixture 1 is 1.6~2.4 mM (which can be 1.8 mM, 1.9 mM, 2.1 mM or 2.2 mM).

[0027] The content of the oxidized conotoxin backbone in the mixture 2 is 8~12 mM (which can be 9 mM, 9.5 mM, 10.5 mM or 11 mM).

[0028] The pyroglutamic acid content in the mixture 2 is 9.6~14.4 mM (which can be 10 mM, 11 mM, 13 mM or 14 mM).

[0029] The sodium hexametaphosphate content in the mixture 2 is 4~6 mM (which can be 4.5 mM, 4.9 mM, 5.1 mM or 5.5 mM).

[0030] The magnesium chloride content in the mixture 2 is 4~6 mM (which can be 4.5 mM, 4.9 mM, 5.1 mM or 5.5 mM).

[0031] The ATP content in the mixture 2 is 0.8~1.2 mM (which may be 0.85 mM, 0.9 mM, 1.1 mM or 1.15 mM).

[0032] The content of the pyroglutamic conotoxin backbone in the mixture 3 is 8~12 mM (which can be 9 mM, 9.5 mM, 10.5 mM or 11 mM).

[0033] The concentration of concentrated ammonia in the mixture 3 is 12-18 mM (which can be 13 mM, 14 mM, 16 mM or 17 mM).

[0034] The stirring temperature is 24~36℃ (it can be 26℃, 28℃, 32℃ or 34℃);

[0035] The acid is hydrochloric acid;

[0036] The purification and separation process includes the steps of precipitation, centrifugation, ammonium sulfate precipitation or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography.

[0037] The content of the thiol oxidase and the lactate dehydrogenase in the mixture 1 is 1600~2400 U (which can be 1800 U, 1900 U, 2100 U or 2200 U).

[0038] The pyroglutamate ligase and the ATP regenerator in the mixture 1 are each in the range of 1600~2400U (which can be 1800 U, 1900 U, 2100 U or 2200 U).

[0039] The ammonia-transferase is 2400~3600 U (it can be 2800 U, 2900 U, 3100 U or 3200 U).

[0040] This application also provides a method for preparing conospirin, including:

[0041] Buffer solution, conopeptide backbone, pyruvate, NAD + Pyroglutamic acid, sodium hexametaphosphate, ATP, magnesium chloride, and concentrated ammonia are mixed and the pH is adjusted to 7.0-9.0 (it can be 7.5, 7.9, 8.0, 8.1, or 8.5). Then, the mixture is mixed with immobilized enzyme to obtain a mixture. The mixture is stirred and the pH is maintained at 7.0-8.5 (it can be 7.4, 7.6, 8.0, or 8.2). The mixture is then separated and purified to obtain conotoxin.

[0042] The immobilized enzyme is the immobilized enzyme described above or the immobilized enzyme prepared by the above method.

[0043] In some specific embodiments of this application, the buffer solution in the above-mentioned method for preparing conospirin is 0.8~1.2 L (which can be 0.9 L, 0.95 L, 1.05 L or 1.1 L), 20~30 mM (which can be 23 mM, 24 mM, 26 mM or 27 mM), pH 7.5~8.5 (which can be 7.8, 7.9, 8.1 or 8.2), and tris(hydroxymethyl)aminomethane hydrochloric acid;

[0044] The content of the conotoxin backbone in the mixture is 8~12 mM (which can be 9 mM, 9.5 mM, 10.5 mM or 11 mM).

[0045] The pyruvate content in the mixture is 26.4~39.6 mM (which can be 28 mM, 32 mM, 35 mM or 37 mM).

[0046] The NAD + The content in the mixture is 1.6~2.4 mM (which can be 1.8 mM, 1.9 mM, 2.1 mM or 2.2 mM).

[0047] The pyroglutamic acid content in the mixture is 9.6~14.4 mM (it can be 10 mM, 11 mM, 13 mM or 14 mM).

[0048] The sodium hexametaphosphate content in the mixture is 4~6 mM (which can be 4.5 mM, 4.9 mM, 5.1 mM or 5.5 mM).

[0049] The magnesium chloride content in the mixture is 4~6 mM (which can be 4.5 mM, 4.9 mM, 5.1 mM or 5.5 mM).

[0050] The ATP content in the mixture is 0.8~1.2 mM (which may be 0.85 mM, 0.9 mM, 1.1 mM or 1.15 mM).

[0051] The concentration of concentrated ammonia in the mixture is 12-18 mM (it can be 13 mM, 14 mM, 16 mM or 17 mM).

[0052] The stirring temperature is 28~42℃ (it can be 30℃, 34℃, 36℃ or 40℃);

[0053] The purification and separation process includes the steps of precipitation, centrifugation, ammonium sulfate precipitation or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography.

[0054] In some specific embodiments of this application, the conospirol backbone of the above-mentioned conospirol preparation method is synthesized by liquid-phase synthesis based on Fmoc-Arg(Pbf)-Cys(Trt)-Cys(Trt)-OH, Fmoc-Asp(OtBu)-His(Trt)-Ala-OH, Fmoc-Trp(Boc)-Cys(Trt)-Arg(Pbf)-OH, Fmoc-Ser(OtBu)-Ser(OtBu)-Lys(Boc)-OH, Fmoc-Lys(Boc)-Gly-Cys(Trt)-OH, Fmoc-Asn(Trt)-Gly-Pro-OH and Fmoc-Gly-Cys(Trt)-OH.

[0055] This invention is highly innovative, and conopod peptides have significant potential for bioactive applications. The method proposed in this application for preparing conopod peptides through liquid-phase synthesis combined with enzyme-catalyzed modification is fundamentally different from existing chemical solid-phase synthesis methods. This invention uses seven tripeptide fragments containing protecting groups, coupled together in liquid phase, to form the conopod peptide backbone. It then uses inexpensive and readily available raw materials such as pyruvate, sodium hexametaphosphate, and pyroglutamic acid as initial materials. A self-constructed directed enzyme catalysis system is used to achieve highly accurate modification, ultimately yielding a structurally complete conopod peptide product. This technical approach overcomes the bottlenecks of traditional solid-phase synthesis, which suffers from numerous byproducts and difficulties in removing protecting groups. The entire process employs environmentally friendly enzymatic reactions, avoiding the use of toxic reagents. Currently, no commercially available conopod peptide preparation process utilizes this liquid-phase-enzyme synergistic strategy. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0057] Figure 1 This application illustrates the synthetic route;

[0058] Figure 2 This application provides information on the properties of the enzymes involved.

[0059] Figure 3 The synthetic reaction formula of Example 1 of this application is shown;

[0060] Figure 4 This shows the HPLC chromatogram of the conospirin backbone after the reaction endpoint in Example 1 of this application;

[0061] Figure 5 The synthetic reaction formula of Example 2 of this application is shown;

[0062] Figure 6 This application shows the liquid HPLC chromatogram of the oxidized conopod peptide backbone after the reaction endpoint in Example 2 of this application;

[0063] Figure 7 The mass spectrum (MS) of the oxidized conotoxin product of Example 2 of this application is shown.

[0064] Figure 8 The synthetic reaction formula of Example 3 of this application is shown;

[0065] Figure 9 The synthetic reaction formula of Example 4 of this application is shown;

[0066] Figure 10 The final product of this application, conopeptide, is shown in the liquid chromatography (HPLC) spectrum (96%).

[0067] Figure 11 The mass spectrometry (MS) spectra of the final product conotoxin in Example 4 of this application are shown. In the positive mode, m / z 595 is the peak of the quaternary charge molecular ion, m / z 792.75 is the peak of the trivalent charge molecular ion, and m / z 1189 is the peak of the divalent charge molecular ion. The molecular weight is 2378.

[0068] Figure 12 The synthetic reaction formula of Example 5 of this application is shown;

[0069] Figure 13 The synthetic reaction formula of Example 6 of this application is shown;

[0070] Figure 14 The synthetic reaction formulas of the comparative examples of this application are shown. Detailed Implementation

[0071] This application discloses a chemical enzyme fusion process for preparing conospirin. 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 can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this invention.

[0072] In some embodiments, conospiropeptide is prepared using seven tripeptide fragments containing protecting groups as raw materials. Through stepwise liquid-phase coupling, using a HATU / DMF system and progressive deprotection, an unmodified conospiropeptide backbone is formed. Subsequently, the backbone is synergistically catalyzed by a YLOxidase and NspLDH oxidase system to generate an intermediate containing an oxidized group. This intermediate is then catalyzed by YLpLigase and BfaPPK to complete N-terminal pyroglutamylation modification, forming a pyroglutaminated conospiropeptide backbone. Finally, the side chain amino groups are amidated by YLAmid enzyme catalysis to ultimately generate the target conospiropeptide. The above enzyme systems have all significantly improved their catalytic activity, substrate specificity, and thermal stability through multi-redirection evolution technology, thereby achieving efficient and continuous enzymatic synthesis. This effectively avoids the problems of cumbersome protecting group operations and numerous byproducts in traditional methods, significantly improving product purity and production efficiency.

[0073] This invention innovatively combines green chemistry and enzymatic methods, utilizing liquid-phase synthesis to efficiently construct the conopod peptide backbone, avoiding the cumbersome protecting group steps of solid-phase methods. Furthermore, a directionally evolved enzyme system, with its high specificity, precisely completes modification and assembly, significantly reducing the risk of mismatch and achieving accurate sequence assembly. This method greatly simplifies the synthesis process, reduces production costs, and simultaneously improves product purity and yield, providing an efficient and environmentally friendly continuous production path for cosmetic-grade conopod peptides. It effectively solves the core problems of low efficiency, heavy pollution, and high cost associated with traditional processes.

[0074] The chemical and enzymatic route for the preparation of conopod peptides in this invention is described in [reference needed]. Figure 1 .

[0075] The relevant information regarding the enzymes involved in this application is as follows.

[0076] Pyroglutamate ligase (YLpLigase): Derived from Streptomyces poonensis (Uniprot ID: A0A918UXJ5), this natural enzyme (WTYLpLigase) has weak activity against conotoxin. After systematic modification (YLpLigase), its activity and expression level were improved. The specific mutation sites are: D9T, H42F, P43A, A68V, P126N, N140I, S141M, V154L, D235H, L252Y, F256S, D260Q, Q280K, H355C.

[0077] Thiol oxidase (YLOxidase): Rhodococcus sp. (Uniprot ID: A0A1X0UCZ9), this natural enzyme (WTYLOxidase) has very weak activity against the conopod peptide backbone. After systematic modification, its expression and activity were improved. The specific mutation sites are: R15G, V18S, T23L, E90Q, W119I, L124D, R149H, N190T, A197M, F226G, P258N, F290L, R292E.

[0078] Lactate dehydrogenase (NspLDH): Nostoc sp. (Uniprot ID: A0A367PX33), this natural enzyme (WTNspLDH) has good NAD content. + It has regenerative capacity, but its expression level and stability are not ideal; through modification of the enzyme (NspLDH), its performance is significantly improved, and its mutation sites are: K95D, R125N, K151L, I152F, D153H.

[0079] ATP regenerator (BfaPPK): Bacteroides faecichinchillae (Uniprot ID: A0A1M5CMU2). The natural enzyme (WTBfaPPK) has decent ATP regeneration activity, but its expression level and stability are not ideal. Through design, the mutant enzyme (BfaPPK) has significantly improved performance. Its specific mutation sites are: S66I, F79T, R126Q, H143A, and Q144C.

[0080] Aminotransferase (YLAmid): Pseudoalteromonas ulvae (Uniprot ID: A0A2C9ZZN4) has low substrate activity and low expression levels. Through systematic experimental optimization and modification, a mutant enzyme (YLAmid) with improved performance in all aspects was finally obtained. The specific mutation sites are: W118S, E123G, F124V, E126T, Y179M, L185A, S224N, Q349F, R455A, C458H, T497I, I610R, and N611L.

[0081] The sequence information of the enzymes involved in this application is shown in Tables 1 and 2.

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086]

[0087] For information on the enzyme properties involved in this application, please refer to [link / reference needed]. Figure 2 .

[0088] The enzymes involved in this application are all produced by laboratory fermentation. The following is the basic operational procedure for preparing the enzyme. First, the gene sequence corresponding to the enzyme was synthesized by a gene company (Anhui General Biotechnology). Then, it was subcloned into the pET28a plasmid through the NdeI / XhoI restriction site. The plasmid was then transformed into E. coli (BL21) cells (Qingke Biotechnology) for plate culture. Finally, single colonies were selected for liquid-scale scale-up culture. The following is the basic procedure for cell scale-up culture: First, single colonies on the plate were transferred to 5 mL of LB medium containing 50 μM kanamycin (37℃) for culture. When the cells grew to the logarithmic growth phase, they were inoculated into 250 mL of LB medium containing the same antibiotic. Finally, they were transferred to a 5 L fermenter for culture. When the cell OD reached 20, 0.5 mM isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added and the protein expression was induced at 28℃ for 8 hours. Then, the cells were centrifuged (4000 rpm, 15 min) to collect 25-35 grams of wet cells. To verify enzyme expression, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0), followed by cell disruption using a freeze-thaw method. After high-speed centrifugation, the supernatant was run on an SDS-PAGE protein gel (sodium dodecyl sulfonate-polyacrylamide gel) to confirm soluble protein expression. The remaining cells, after confirmation, were mixed with buffer (10 g of wet cells to approximately 200 mL of the above buffer), then subjected to high-pressure cell disruption and high-speed centrifugation (16000 rpm, 10 min) to remove the cell wall. The resulting enzyme-containing supernatant was used directly (the liquid enzyme activity is 300–1500 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or further purified and immobilized for use (in solid enzyme reactions). LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0089] The enzyme immobilization process described in this application is as follows: ammonium sulfate solid is added incrementally to the crude enzyme solutions of pyroglutamate ligase (YLpLigase), thiol oxidase (YLOxidase), lactate dehydrogenase (NspLDH), ATP regenerase (BfaPPK), and aminotransferase (YLAmid) until enzyme precipitation (40%–60%, w / v ammonium sulfate / buffer). The enzyme solid is then collected by centrifugation (10,000 rpm, 15 min) and slowly dissolved in 25 mM pH 8.0 Tris buffer. Finally, it is desalted using a G25 size exclusion column (purchased from Sigma) and separated using a DEAE Seplite FF anion exchange column (Xi'an Lanxiao Company) to obtain pre-purified liquid enzymes YLpLigase, YLOxidase, NspLDH, BfaPPK, and YLAmid. In the immobilized mixed enzyme, the above-mentioned initially purified enzyme was immobilized using LX-1000 EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of 2:2:2:2:3. The basic immobilization method was as follows: 10,000 U of the mixed enzyme mixed according to the above activity unit ratio was dissolved in 2 L of 50 mM pH 8.0 potassium phosphate solution, followed by the addition of phenoxyacetic acid (final concentration 60 mM) and 900 g 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 finally washed three times each with distilled water and 25 mM pH 8.0 phosphate buffer, and then dried at low temperature for later use. The YLpLigase / YLOxidase / NspLDH / BfaPPK / YLAmid immobilized mixed enzyme had 75%~92% of the activity of the corresponding liquid enzyme.

[0090] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this application are all commercially available products and can be purchased from the market.

[0091] The present invention will be further illustrated below with reference to the embodiments.

[0092] Example 1: Preparation of conopeptide backbone using tripeptide fragments as raw materials via liquid-phase synthesis.

[0093] See the synthesis reaction formula. Figure 3 .

[0094] (1) Resin bonding steps:

[0095] Wang resin (16.7 g, 10 mmol) with a substitution degree of 0.60 mmol / g was added to the reactor, and DMF was added to swell the resin for 30 min, followed by filtration. In another 250 mL beaker, Fmoc-Arg(Pbf)-Cys(Trt)-Cys(Trt)-OH (26.8 g, 20 mmol) was dissolved in 80 mL of DMF. The mixture was cooled to 0–5 °C, and DIEA (5.2 mL, 30 mmol), DIC (2.78 g, 22 mmol), and DMAP (244 mg, 2.0 mmol) were added sequentially. The mixture was stirred for 5 min at 0–5 °C and then poured into the reactor. The reaction was allowed to proceed at room temperature for 16 h. The mixture was then filtered, washed three times with 60 mL of DMF, and then de-Fmoc was removed twice with 20% piperidine / DMF solution (10 min + 10 min). The mixture was then filtered, washed six times with DMF, and finally filtered again.

[0096] (2) Coupling and Deprotection Steps

[0097] Weigh 17.2 g (20 mmol), HOBt (2.97 g, 22 mmol), and DIEA (5.2 mL, 30 mmol) into a 250 mL beaker. Add 80 mL of DMF and stir at 0–5 °C until completely dissolved. Add 2.78 g (22 mmol) of DIC and stir at 0–5 °C for 5 min. Pour the mixture into a reactor. React at room temperature for 1.5 h. After the reaction is complete as determined by ninhydrin assay, filter the mixture and wash three times with 60 mL of DMF. Remove Fmoc twice with 20% piperidine / DMF solution (10 min + 10 min). Remove the solvent under reduced pressure, wash six times with DMF, and then remove the solvent under reduced pressure.

[0098] Repeat the above coupling and deprotection steps to sequentially condense and protect the tripeptides Fmoc-Trp(Boc)-Cys(Trt)-Arg(Pbf)-OH, Fmoc-Ser(OtBu)-Ser(OtBu)-Lys(Boc)-OH, Fmoc-Lys(Boc)-Gly-Cys(Trt)-OH, Fmoc-Asn(Trt)-Gly-Pro-OH, and Fmoc-Gly-Cys(Trt)-Cys(Trt)-OH. Specifically, the coupling time for Fmoc-Asn(Trt)-Gly-Pro was 5 h. After all amino acid coupling was complete, methanol was added to shrink the resin, finally obtaining the fully protected fragment resin.

[0099] Fmoc-Gly-Cys(Trt)-Cys(Trt)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Cys(Trt)-Ser(OtBu)-Ser(OtBu)-Ly s(Boc)-Trp(Boc)-Cys(Trt)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Trt)-Cys(Trt)-Wang Resin

[0100] (3) Pyrolysis step:

[0101] Prepare a lysis buffer (TFA:TIS:water = 95:2.5:2.5) at a ratio of 10 mL / g peptide resin, mix thoroughly, and cool to 0–5 °C. Place the peptide resin in a 250 mL round-bottom flask, add the lysis buffer at 0–5 °C, and then maintain the temperature at 0–5 °C with stirring for 3 h. After the reaction is complete, filter under vacuum, wash the resin three times with DCM, and collect the filtrate. Concentrate under reduced pressure at 40–45 °C to remove TFA, and then slowly add the distillation residue dropwise to a large amount of methyl ether at 0–5 °C to precipitate a white solid. After the addition is complete, continue stirring at 0–5 °C for 30 min, filter, wash three times with methyl ether, and collect the filter cake.

[0102] The filter cake was vacuum dried at 25°C to constant weight, and 15.9 g of conotoxin backbone product was collected, with a yield of 73.3%. Liquid chromatography-HPLC analysis confirmed that it was conotoxin backbone. Figure 4 Store in a sealed container at -20°C for later use.

[0103] Example 2: Preparation of oxidized conospiropeptide backbone using conospiropeptide backbone as raw material via liquid enzyme (YLOxidase, NspLDH) oxidation.

[0104] See the synthesis reaction formula. Figure 5 .

[0105] Add 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), and 1.3 g of NAD to 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution. +After adding 2 mM, the pH of the solution was adjusted back to 8.0. Then, 2000 U of crude YLOxidase enzyme solution and 2000 U of crude NspLDH enzyme solution were added at once to start the reaction. After stirring gently at 30°C for 3 hours, hydrochloric acid was added to terminate the reaction. The protein was precipitated and centrifuged to remove the protein. Subsequently, the target peptide was initially enriched by ammonium sulfate precipitation or organic solvent precipitation. Then, it was further purified by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. Then, high-resolution purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic difference. Finally, 20.7 g of oxidized conopodyne white solid was obtained by drying (final yield 91%). After the purified product was detected by HPLC and mass spectrometry (MS), it was lyophilized and stored at -80°C or in a vacuum desiccator. Low temperature operation was maintained throughout the process to ensure stability. The detection results are shown in the figure. Figure 6 (HPLC) Figure 7 (Mass spectrometry).

[0106] Example 3: Preparation of pyroglutamic conospiroid backbone using oxidized conospiroid peptide backbone as raw material and liquid enzymes (YLpLigase, BfaPPK)

[0107] See the synthesis reaction formula. Figure 8 .

[0108] Add 22.7 g of oxidized conopodyl peptide backbone (10 mM), 1.5 g of pyroglutamic acid (12 mM), 3.1 g of sodium hexametaphosphate (5 mM), 1.0 g of magnesium chloride hexahydrate (5 mM), and 0.6 g of ATP (1 mM) to 1 L of 25 mM pH 8.0 Tris.HCl solution. Adjust the pH of the solution back to 8.0, then add 2000 U of YLpLigase crude enzyme solution and 2000 U of... The reaction was initiated with crude BfaPPK enzyme solution; the reaction solution was gently stirred at 30°C, and the pH of the reaction system was maintained between 7.5 and 9.0 using acid and base during the reaction; after 4 hours of reaction, hydrochloric acid was added to terminate the reaction, and the protein was removed by precipitation and centrifugation. Subsequently, the target peptide was initially enriched by ammonium sulfate precipitation or organic solvent precipitation; then, it was further purified by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight; then, high-resolution purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic difference; finally, 21.0 g of pyroglutamic conopod peptide backbone white solid was obtained by drying (final yield 89%); it was lyophilized and stored at -80°C or in a vacuum desiccator, with low temperature operation throughout to ensure stability.

[0109] Example 4: Preparation of conospiropeptide using pyroglutamic conospiropeptide backbone as raw material and catalyzed by liquid enzyme (YLAmid)

[0110] See the synthesis reaction formula. Figure 9 .

[0111] 23.8 g of pyroglutaminated conopodyl peptide backbone (10 mM) and 1.1 mL of concentrated ammonia (15 mM) were added to 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. The pH was then adjusted back to 8.0, and 3000 U of crude YLAmid enzyme solution was added in one step to initiate the reaction. The reaction solution was gently stirred at 30°C for 2 hours until the reaction was complete. The enzyme was precipitated by acid addition, and the precipitate was removed by centrifugation (~10000 rpm, 10 min). The target peptide was then initially enriched by ammonium sulfate precipitation or organic solvent precipitation. Further purification was then performed by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. High-resolution purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic differences. Finally, 20.4 g of conopodyl peptide white solid product was obtained by drying (final yield 86%). The purified product was confirmed by HPLC and mass spectrometry (MS). See [link to relevant documentation]. Figure 10 , Figure 11 Freeze-dried products should be stored at -80°C or in a vacuum desiccator, with low-temperature operation throughout to ensure stability.

[0112] Example 5: Preparation of conospiropeptide from conospiropeptide backbone using liquid enzymes (YLOxidase, NspLDH, YLpLigase, BfaPPK, YLAmid) in a single conversion.

[0113] See the synthesis reaction formula. Figure 12 .

[0114] Add 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), and 1.3 g of NAD to 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. +The solution was prepared by adding 2 mM sodium pyroglutamate (12 mM), 3.1 g sodium hexametaphosphate (5 mM), 0.6 g ATP (1 mM), 1.0 g magnesium chloride hexahydrate (5 mM), and 1.1 mL concentrated ammonia (15 mM) to adjust the pH back to 8.0. Then, 2000 U of crude YLOxidase, 2000 U of crude NspLDH, 2000 U of crude YLpLigase, 2000 U of crude BfaPPK, and 3000 U of crude YLAmid were added at once to initiate the reaction. The reaction solution was gently stirred at 30°C, and the pH of the reaction system was maintained between 7.0 and 9.0 using acid and alkali during the reaction. After 6 hours, the reaction was completed, and the enzymes in the reaction solution were precipitated by adding acid and then rapidly centrifuged (~10000). The enzyme precipitate was removed by 10 min at 10 rpm, followed by ammonium sulfate precipitation or organic solvent precipitation to initially enrich the target peptide. Further purification was then performed by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine molecular weight. High-resolution purification was then achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic differences. Finally, drying yielded 19.4 g of conostin as a white solid (final yield 82%). The purity of the purified product was determined by HPLC and mass spectrometry (MS), and then lyophilized and stored at -80°C or in a vacuum desiccator, ensuring stability throughout the process.

[0115] Example 6: Using conospirin backbone and NAD + Conospirin was prepared by a one-time conversion of immobilized mixed enzymes using ATP and other raw materials.

[0116] See the synthesis reaction formula. Figure 13 .

[0117] The reaction is similar to that in Example 5 above, but an immobilized enzyme is used, so it can be recycled multiple times.

[0118] Add 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), and 1.3 g of NAD to 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. +The solution was prepared by adding 2 mM sodium pyroglutamate (12 mM), 3.1 g sodium hexametaphosphate (5 mM), 0.6 g ATP (1 mM), 1.0 g magnesium chloride hexahydrate (5 mM), and 1.1 mL concentrated ammonia (15 mM) to adjust the pH of the solution back to 8.0. Then, 10,000 U of immobilized mixed enzyme was added in a single batch to initiate the reaction. The reaction was carried out at 35°C with gentle stirring, maintaining the pH at approximately 7.0–8.5 throughout the reaction. After 8 hours, the reaction was complete, and the immobilized mixed enzyme was collected by filtration (the enzyme retained 88% of its initial activity after 6 uses). The target peptide was initially enriched by ammonium sulfate precipitation or organic solvent precipitation. Further purification was achieved by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. High-resolution purification was then achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic differences. Finally, 20.6 g of conopeptide white solid was obtained by drying (final yield 87%). After the purity of the purified product was detected by HPLC and mass spectrometry (MS), it was lyophilized and stored at -80℃ or in a vacuum desiccator. The entire process was carried out at low temperature to ensure stability.

[0119] Comparative Example: Conospirin was prepared in a single-step conversion using conospirin backbone as raw material and liquid enzymes (WTYLOxidase, WTNspLDH, WTYLpLigase, WTBfaPPK, WTYLAmid).

[0120] See the synthesis reaction formula. Figure 14 .

[0121] Similar to Example 5 above, each enzyme was replaced with the natural enzyme WT.

[0122] Add 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), and 1.3 g of NAD to 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. +The solution was prepared by adding 2 mM sodium pyroglutamate (12 mM), 3.1 g sodium hexametaphosphate (5 mM), 0.6 g ATP (1 mM), 1.0 g magnesium chloride hexahydrate (5 mM), and 1.1 mL concentrated ammonia (15 mM) to adjust the pH back to 8.0. Then, 2000 U WTYLOxidase crude enzyme solution, 2000 U WTNspLDH crude enzyme solution, 2000 U WTYLpLigase crude enzyme solution, 2000 U WTBfaPPK crude enzyme solution, and 3000 U WTYLAmid crude enzyme solution were added at once to start the reaction. The reaction solution was gently stirred at 30°C, and the pH of the reaction system was maintained between 7.0 and 9.0 using acid and alkali during the reaction. After 6 hours, the reaction was completed, and the enzymes in the reaction solution were precipitated by adding acid and then rapidly centrifuged (~10000). The enzyme precipitate was removed by 10 min at 10 rpm, followed by ammonium sulfate precipitation or organic solvent precipitation to initially enrich the target peptide. Further purification was then achieved by ion exchange chromatography (IEX) based on charge difference, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine molecular weight. High-resolution purification was then achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) based on hydrophobic differences. Finally, 4.1 g of conospirin was dried to obtain a white solid (final yield 17%). The purity of the purified product was determined by HPLC and mass spectrometry (MS), and then lyophilized and stored at -80°C or in a vacuum desiccator, ensuring stability throughout the process.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. An enzyme combination, characterized in that, It consists of a pyroglutamate ligase with the amino acid sequence SEQ ID NO: 1, a thiol oxidase with the amino acid sequence SEQ ID NO: 2, an aminotransferase with the amino acid sequence SEQ ID NO: 3, a lactate dehydrogenase with the amino acid sequence SEQ ID NO: 4, and an ATP regenerator with the amino acid sequence SEQ ID NO:

5.

2. A method for preparing immobilized enzymes, characterized in that, include: The pyroglutamate ligase with amino acid sequence SEQ ID NO: 1, the thiol oxidase with amino acid sequence SEQ ID NO: 2, the ammonia-transferase with amino acid sequence SEQ ID NO: 3, the lactate dehydrogenase with amino acid sequence SEQ ID NO: 4, and the ATP regenerator with amino acid sequence SEQ ID NO: 5 were mixed to obtain a mixed enzyme. The mixed enzyme was dissolved in potassium phosphate solution, then mixed with phenoxyacetic acid and epoxy resin, stirred, and filtered to obtain immobilized enzyme; The pyroglutamate ligase, the thiol oxidase, the lactate dehydrogenase, the ATP regenerator, and the ammonylase are mixed in a ratio of 2:2:2:2:3, and the enzyme activity of the mixed enzyme is 10,000 U. The potassium phosphate solution has a concentration of 50 mM, a pH of 8.0, and a volume of 2 L. The final concentration of the phenoxyacetic acid is 60 mM; The epoxy resin is LX-1000 EP epoxy resin, with a mass of 900 grams; The stirring time is 8 hours.

3. The immobilized enzyme prepared by the method described in claim 2.

4. The application of the enzyme combination as described in claim 1 or the immobilized enzyme as described in claim 3 in the preparation of conopodeptides, characterized in that, Using conospirin backbone as raw material, the amino acid sequence of the conospirin backbone is Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys.

5. A method for preparing conospirin, characterized in that, include: Buffer, conopeptide backbone, pyruvate and NAD + Mix, adjust pH to 7.0-9.0, then mix with thiol oxidase with amino acid sequence SEQ ID NO: 2 and lactate dehydrogenase with amino acid sequence SEQ ID NO: 4 to obtain mixture 1. Stir, mix with acid, separate and purify to obtain oxidized conospirin backbone; The buffer solution, oxidized conospirin backbone, pyroglutamic acid, sodium hexametaphosphate, magnesium chloride and ATP were mixed and the pH was adjusted to 7.0-9.

0. Then it was mixed with pyroglutamic acid ligase with amino acid sequence SEQ ID NO: 1 and ATP regenerase with amino acid sequence SEQ ID NO: 5 to obtain mixture 2. The mixture was stirred and the pH was maintained at 7.5-9.

0. It was then mixed with acid, separated and purified to obtain pyroglutaminated conospirin backbone. Mix the buffer solution, the pyroglutamic conospirin backbone, and concentrated ammonia to obtain mixture 3. Adjust the pH to 7.0-9.0, then mix with the ammonia-transferase with the amino acid sequence SEQ ID NO: 3, stir, mix with acid, separate and purify to obtain conospirin; The amino acid sequence of the conospirin backbone is Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys.

6. The preparation method according to claim 5, characterized in that, The buffer solution is 1 L 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid; The content of the conospiro peptide backbone in mixture 1 is 10 mM; The content of pyruvate in mixture 1 is 33 mM; The NAD + The content in mixture 1 is 2 mM; The content of the oxidized conospirin backbone in the mixture 2 is 10 mM; The pyroglutamic acid content in mixture 2 is 12 mM; The sodium hexametaphosphate content in mixture 2 is 5 mM; The magnesium chloride content in mixture 2 is 5 mM; The ATP content in mixture 2 is 1 mM; The content of the pyroglutamic conospiro peptide backbone in the mixture 3 is 10 mM; The concentration of concentrated ammonia in mixture 3 is 15 mM; The stirring temperature is 30°C; The acid is hydrochloric acid; The purification and separation process includes the steps of precipitation, centrifugation, ammonium sulfate precipitation or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography. The content of the thiol oxidase and the lactate dehydrogenase in the mixture 1 is 2000 U each; The content of the pyroglutamate ligase and the ATP regenerase in the mixture 2 is 2000 U each; The ammonia-transferase is 3000 U.

7. A method for preparing conospirin, characterized in that, include: Buffer solution, conopeptide backbone, pyruvate, NAD + Pyroglutamic acid, sodium hexametaphosphate, ATP, magnesium chloride, and concentrated ammonia were mixed and the pH was adjusted to 7.0-9.

0. Then, the mixture was mixed with immobilized enzyme to obtain a mixture. The mixture was stirred and the pH was maintained at 7.0-8.

5. After separation and purification, conotoxin was obtained. The immobilized enzyme is the immobilized enzyme according to claim 3; The amino acid sequence of the conospirin backbone is Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys.

8. The preparation method according to claim 7, characterized in that, The buffer solution is 1 L 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid; The content of the conospiro peptide backbone in the mixture is 10 mM; The content of pyruvate in the mixture is 33 mM; The NAD + The content in the mixture is 2 mM; The pyroglutamic acid content in the mixture is 12 mM; The sodium hexametaphosphate content in the mixture is 5 mM; The magnesium chloride content in the mixture is 5 mM; The ATP content in the mixture is 1 mM; The concentration of concentrated ammonia in the mixture is 15 mM. The stirring temperature is 35°C; The purification and separation process includes the steps of precipitation, centrifugation, ammonium sulfate precipitation or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography.

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