Enzyme mutant and application thereof in preparation of conopeptide

Abstract

The invention relates to the technical field of biochemistry, in particular to an enzyme mutant and application thereof in preparation of conopeptide. According to the preparation method, a strategy of combining green chemistry and an enzyme method is adopted, seven tripeptide fragments are taken as raw materials, a conopeptide main chain is prepared through a liquid-phase synthesis method, and then enzymatic precise modification and synthesis are realized by utilizing a multiple directed evolution enzyme concerted catalysis system. Based on the efficient connection characteristic of a liquid-phase synthesis system and the high substrate specificity of an enzymatic system, the complex protection group operation in traditional solid-phase synthesis is effectively avoided, generation of by-products is remarkably reduced, meanwhile, fixed-point modification and precise assembly of a conopeptide main chain are achieved, and the application prospect is wide. And an efficient and green new path is provided for the continuous production of the cosmetic-grade high-purity conopeptide.

Description

Technical Field

[0001] The present application relates to the field of biochemical technology, and in particular to the preparation of cono peptides by a chemical enzyme fusion process. Background Art

[0002] Conus peptide, a neuromodulatory peptide extracted from the toxin of the marine cone snail, possesses both potent anti-wrinkle and neuroprotective properties. Its mechanism of action is to precisely block nerve signaling, inhibiting excessive muscle contraction and thereby reducing the formation of dynamic wrinkles. It also activates skin repair pathways, promoting collagen and elastin regeneration, and strengthening the skin's structural support. In cosmetic applications, conus peptide effectively relaxes dynamic facial muscles by regulating calcium channels at the neuromuscular junction, rapidly smoothing deep expression lines such as glabellar lines and creases. Its anti-wrinkle effects last for over 48 hours, combining immediate smoothing with long-lasting firming. Furthermore, by inhibiting the release of inflammatory factors, conus peptide alleviates skin sensitivity and redness, and enhances skin barrier function. Due to its unique synergistic effects of neuromodulation and skin regeneration, conus peptide has significant potential for application in anti-aging skincare, medical aesthetic anti-wrinkle treatments, and the repair of neurological skin problems. It is particularly suitable for high-end skincare products that require both soothing and firming benefits.

[0003] Currently available methods for preparing cone snail peptides include chemical synthesis, biosynthesis based on recombinant DNA technology, and direct extraction from natural cone snails. For example, a linear cone snail peptide precursor is synthesized by solid-phase synthesis using Rink resin as a carrier, followed by the stepwise coupling of amino acids modified with Fmoc protecting groups. After cleavage and deprotection with a mixture of phenol / dithioglycolic acid / TFA, the peptide is properly folded to form disulfide bonds, ultimately yielding a biologically active cone snail peptide (CN1120174 C-Preparation Method for Marine Cone Snail Analgesic Peptide). Shenzhen BGI Genomics extracts crude venom from barrel-shaped cone snail venom tubes. After extraction with a solution containing 30% acetonitrile and 0.1% trifluoroacetic acid, the target peptide is enriched through reductive alkylation and solid-phase extraction column chromatography. It is then purified by high-performance liquid chromatography and its amino acid sequence identified, ultimately yielding the active cone snail toxin κ-CPTx-btl01 (CN 107074909 B-Conotoxin Peptide κ-CPTx-btl01, Preparation Method, and Application). Alternatively, a recombinant vector containing a GST tag and an enterokinase recognition sequence can be constructed through genetic recombination technology, and the fusion protein can be expressed in Escherichia coli. High-purity μ-conotoxin can be obtained through GST affinity chromatography purification, enterokinase cleavage, and ion exchange chromatography (CN 116355932 B - Recombinant vector and method for preparing μ-conotoxin).

[0004] However, the solid-phase synthesis method requires repeated protective group operations, resulting in a cumbersome process, many by-products, and reliance on toxic solvents, causing serious environmental pollution; the liquid-phase step-by-step method is limited by multi-step intermediate synthesis and purification, resulting in low efficiency and high cost; the enzymatic ligation method is prone to producing sequence-mismatched by-products due to the limited substrate compatibility of the enzyme; and recombinant protein technology faces bottlenecks such as low expression levels and complex purification processes. Summary of the Invention

[0005] In view of this, the present application provides a chemical-enzyme fusion process for preparing cono peptides, which adopts a green chemistry and enzymatic combination strategy, uses seven tripeptide fragments such as Fmoc-Arg(Pbf)-Cys(Trt)-Cys(Trt)-OH, Fmoc-Asp(OtBu)-His(Trt)-Ala-OH as raw materials, prepares the cono peptide backbone by liquid phase synthesis, and then uses a multi-redirected evolutionary enzyme synergistic catalytic system to achieve enzymatic precision modification and synthesis; based on the efficient connection 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 the generation of by-products, and at the same time realizes the site-specific modification and precise assembly of the cono peptide backbone, providing a new efficient and green path for the continuous production of cosmetic-grade high-purity cono peptides.

[0006] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0007] The present application provides any one of a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1, a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2, an aminase having an amino acid sequence of SEQ ID NO: 3, a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5.

[0008] The present application also provides an enzyme combination, comprising at least two of a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1, a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2, an aminase having an amino acid sequence of SEQ ID NO: 3, a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5.

[0009] The present application also provides an immobilized enzyme comprising the above enzyme combination.

[0010] The present application also provides a method for preparing the above-mentioned immobilized enzyme, comprising:

[0011] mixing a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1, a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2, an aminase having an amino acid sequence of SEQ ID NO: 3, a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5 to obtain a mixed enzyme;

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

[0013] In some specific embodiments of the present application, the pyroglutamate ligase, the sulfhydryl oxidase, the lactate dehydrogenase, the ATP regenerating enzyme, and the aminase in the above-mentioned method for preparing the immobilized enzyme are mixed according to the ratio of enzyme activity units of 2:(1-3):(1-3):(1-3):(1-3) (which may be 2:2:2:2:2:2.8, 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 may be 7000 U, 9000 U, 11000 U or 13000 U);

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

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

[0016] The epoxy resin is LX-1000 EP epoxy resin, with a mass of 800 to 1000 grams (can be 880 grams, 890 grams, 910 grams or 920 grams);

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

[0018] The present application also provides the use of the above enzyme combination, the above immobilized enzyme or the immobilized enzyme prepared by the above immobilized enzyme preparation method in the preparation of conopeptides.

[0019] The present application also provides a method for preparing conopeptide, comprising:

[0020] Buffer, conopeptide backbone, pyruvate and NAD +Mixing, adjusting the pH to 7.0-9.0 (can be 7.5, 7.9, 8.1 or 8.5), then mixing with sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2 and lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4 to obtain a mixture 1, stirring, mixing with an acid, and separating and purifying to obtain an oxidized conopeptide backbone;

[0021] Mixing a buffer, an oxidized conopeptide backbone, pyroglutamic acid, sodium hexametaphosphate, magnesium chloride, and ATP, adjusting the pH to 7.0-9.0 (which may be 7.5, 7.9, 8.0, 8.1, or 8.5), and then mixing with a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1 and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5 to obtain a mixture 2, stirring, maintaining the pH at 7.5-9.0 (which may be 7.8, 7.9, 8.0, 8.1, or 8.5), mixing with an acid, and separating and purifying to obtain a pyroglutamated conopeptide backbone;

[0022] The buffer solution, the pyroglutamylated cono peptide backbone, and concentrated ammonia solution are mixed, and the pH value is adjusted to 7.0-9.0 (which can be 7.5, 7.9, 8.0, 8.1, or 8.5). The mixture is then mixed with an aminase having an amino acid sequence of SEQ ID NO: 3 to obtain a mixture 3. The mixture is stirred, mixed with an acid, and separated and purified to obtain the cono peptide.

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

[0024] The content of the conopeptide backbone in the mixture 1 is 8-12 mM (can be 9 mM, 9.5 mM, 10.5 mM or 11 mM);

[0025] The content of the pyruvic acid in the mixture 1 is 26.4-39.6 mM (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 (can be 1.8 mM, 1.9 mM, 2.1 mM or 2.2 mM);

[0027] The content of the oxidized conopeptide backbone in the mixture 2 is 8-12 mM (can be 9 mM, 9.5 mM, 10.5 mM or 11 mM);

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

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

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

[0031] The content of ATP in the mixture 2 is 0.8-1.2 mM (can be 0.85 mM, 0.9 mM, 1.1 mM or 1.15 mM);

[0032] The content of the pyroglutaminated conopeptide backbone in the mixture 3 is 8-12 mM (can be 9 mM, 9.5 mM, 10.5 mM or 11 mM);

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

[0034] The stirring temperature is 24-36°C (can be 26°C, 28°C, 32°C or 34°C);

[0035] The acid is hydrochloric acid;

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

[0037] The content of the sulfhydryl oxidase and the lactate dehydrogenase in the mixture 1 is 1600-2400 U (or 1800 U, 1900 U, 2100 U, or 2200 U), respectively;

[0038] The content of the pyroglutamate ligase and the ATP regenerating enzyme in the mixture 1 is 1600-2400 U (can be 1800 U, 1900 U, 2100 U or 2200 U);

[0039] The aminotransferase concentration is 2400-3600 U (can be 2800 U, 2900 U, 3100 U or 3200 U).

[0040] The present application also provides a method for preparing conopeptide, comprising:

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

[0042] The immobilized enzyme is the above-mentioned immobilized enzyme or the immobilized enzyme prepared by the above-mentioned method for preparing the immobilized enzyme.

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

[0044] The content of the conopeptide backbone in the mixture is 8-12 mM (can be 9 mM, 9.5 mM, 10.5 mM or 11 mM);

[0045] The content of the pyruvate in the mixture is 26.4-39.6 mM (can be 28 mM, 32 mM, 35 mM or 37 mM);

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

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

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

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

[0050] The content of ATP in the mixture is 0.8-1.2 mM (can be 0.85 mM, 0.9 mM, 1.1 mM or 1.15 mM);

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

[0052] The stirring temperature is 28-42°C (can be 30°C, 34°C, 36°C or 40°C);

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

[0054] In some specific embodiments of the present application, the main chain of the cono peptide in the above-mentioned method for preparing cono peptide 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)-Cys(Trt)-OH.

[0055] The present invention is quite innovative, and cono peptides have significant biological activity application prospects. The method for preparing cono peptides by combining liquid phase synthesis with enzyme-catalyzed modification proposed in this application is essentially different from the existing chemical solid-phase synthesis method. The present invention uses seven tripeptide fragments containing protective groups to be connected in liquid phase to form the main chain of cono peptides, and then uses cheap bulk products such as pyruvic acid, sodium hexametaphosphate, and pyroglutamic acid as initial raw materials. High-accuracy modification is achieved through an independently constructed directed enzyme catalysis system, and finally a structurally complete cono peptide product is obtained. This technical path breaks through the bottleneck of many by-products and difficult removal of protective groups in traditional solid-phase synthesis. Environmentally friendly enzymatic reactions are used throughout the process, avoiding the use of toxic reagents. However, all cono peptide preparation processes currently on the market do not adopt this liquid phase-enzyme catalysis synergistic strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0057] Figure 1 The synthetic route of this application is shown;

[0058] Figure 2 Show the enzyme property information involved in this application;

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

[0060] Figure 4 1 shows the HPLC spectrum of the conopeptide backbone after the reaction end point of Example 1 of the present application;

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

[0062] Figure 6 The liquid HPLC spectrum of the oxidized conopeptide backbone after the reaction endpoint of Example 2 of the present application is shown;

[0063] Figure 7 The mass spectrum (MS) of the oxidized conopeptide of the product of Example 2 of the present application is shown;

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

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

[0066] Figure 10 The HPLC chromatography (HPLC) of the final product of Example 4 of the present application is shown, and the purity is 96%;

[0067] Figure 11 The mass spectrum (MS) of the final product of Example 4 of the present application is shown, wherein, in the positive mode, m / z 595 is a quadruple-charged molecular ion peak, m / z 792.75 is a triply-charged molecular ion peak, and m / z 1189 is a doubly-charged molecular ion peak, and the molecular weight is 2378;

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

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

[0070] Figure 14 Shown is the synthetic reaction formula of the comparative example of this application. DETAILED DESCRIPTION

[0071] The application discloses a chemoenzyme fusion process for preparing conopeptides, and those skilled in the art can learn from this article content and appropriately improve process parameters to realize. It is particularly important to point out that all similar replacements and modifications are apparent to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the present invention.

[0072] In some embodiments, the preparation of cono peptides uses seven tripeptide fragments containing protecting groups as raw materials. Through liquid phase step-by-step coupling, the HATU / DMF system and gradual deprotection are used to form an unmodified cono peptide backbone. Subsequently, the backbone is catalyzed by the YLOxidase and NspLDH oxidase systems to generate an intermediate containing an oxidized group. This intermediate is then catalyzed by YLpLigase and BfaPPK to complete the N-terminal pyroglutamylation modification to form a pyroglutamated cono peptide backbone. Finally, the side chain amino group is catalyzed by YLAmid enzyme to complete the amidation modification, ultimately generating the target cono peptide. The above enzyme systems have significantly improved their catalytic activity, substrate specificity and thermal stability through multi-redirected evolution technology, thereby achieving efficient and continuous enzymatic synthesis, effectively avoiding the problems of cumbersome protection group operations and numerous by-products in traditional methods, and significantly improving product purity and production efficiency.

[0073] This invention innovatively combines green chemistry with enzymatic methods, utilizing liquid-phase synthesis to efficiently construct the cono peptide backbone, avoiding the tedious protecting group steps of the solid-phase method. The enzyme system, modified by directed evolution, then precisely completes modification and splicing with its high specificity, significantly reducing the risk of mismatches and achieving precise sequence assembly. This method significantly simplifies the synthesis process, reduces production costs, and simultaneously improves product purity and yield. This provides an efficient, environmentally friendly, and continuous production path for cosmetic-grade cono peptides, effectively addressing the core challenges of low efficiency, heavy pollution, and high cost associated with traditional processes.

[0074] The chemical and enzymatic preparation routes of conopeptides of the present invention are shown in Figure 1 .

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

[0076] Pyroglutamate ligase (YLpLigase): Derived from Streptomyces poonensis (UniprotID: A0A918UXJ5), this natural enzyme (WTYLpLigase) has weak activity towards conoside peptides. After system modification (YLpLigase), its activity and expression level have been improved. The specific mutation sites are: D9T, H42F, P43A, A68V, P126N, N140I, S141M, V154L, D235H, L252Y, F256S, D260Q, Q280K, H355C.

[0077] Sulfhydryl oxidase (YLOxidase): Rhodococcus sp. (Uniprot ID: A0A1X0UCZ9). This natural enzyme (WTYLOxidase) has very weak activity on the conopeptide backbone. After system modification (YLOxidase), its expression and activity are 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), the natural enzyme (WTNspLDH) has good NAD + Regeneration ability, but its expression level and stability are not ideal; through the modification of this enzyme (NspLDH), its performance has been significantly improved, and its mutation sites are: K95D, R125N, K151L, I152F, D153H.

[0079] ATP regeneration enzyme (BfaPPK): Bacteroides faecichinchillae (Uniprot ID: A0A1M5CMU2), the natural enzyme (WTBfaPPK) has good ATP regeneration activity, but the expression level and stability are not very ideal; through design, the performance of the mutant enzyme (BfaPPK) is greatly improved, and its specific mutation sites are: S66I, F79T, R126Q, H143A, Q144C.

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

[0081] For the sequence information of the enzymes involved in this application, please refer to Table 1 and Table 2.

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086]

[0087] For information on enzyme properties involved in this application, please refer to Figure 2 .

[0088] The enzymes described in this application are all produced by laboratory fermentation. The following is a basic process for preparing the enzymes. First, the gene sequence corresponding to the enzyme was synthesized by a genetics company (Anhui General Biotechnology). The enzyme was then subcloned into the pET28a plasmid using the NdeI / XhoI restriction sites. This plasmid was then transformed into E. coli (BL21) cells (Qingke Biotechnology) for plate culture. Finally, a single colony was selected for liquid amplification. The following is a basic process for amplification: First, a single colony from a plate was transferred to 5 mL of LB medium containing 50 μM kanamycin (37°C). After the cells reached logarithmic phase, they were inoculated into 250 mL of LB medium containing the same antibiotic and then transferred to a 5 L fermentor for culture. When the cell OD reached ~20, protein expression was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 28°C for 8 hours. The wet cells were then harvested by centrifugation (4000 rpm, 15 minutes) to yield 25-35 grams. To verify enzyme expression, a small amount of cells was mixed evenly with Tris-HCl buffer (50 mM, pH 8.0). Cells were then disrupted by freeze-thaw, centrifuged at high speed, and the supernatant was run on an SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel) to confirm soluble protein expression. Once the cells were confirmed to be correct, the remaining cells were mixed with buffer (10 g of wet cells in approximately 200 mL of the aforementioned buffer), then subjected to high-pressure cell disruption and high-speed centrifugation (16,000 rpm, 10 min) to remove the cell walls. The resulting enzyme-containing supernatant was used directly (the enzyme activity ranged from 300 to 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 solid enzyme reactions). LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0089] The mixed enzyme immobilization process involved in this application is as follows: ammonium sulfate solid is gradually added to crude enzyme solutions of pyroglutamate ligase (YLpLigase), sulfhydryl oxidase (YLOxidase), lactate dehydrogenase (NspLDH), ATP regenerating enzyme (BfaPPK), and aminase (YLAmid) until the enzymes precipitate (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 Tris buffer, pH 8.0. Finally, the solution is desalted using a G25 size exclusion chromatography column (purchased from Sigma) and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the pre-purified liquid enzymes YLpLigase, YLOxidase, NspLDH, BfaPPK, and YLAmid. In the immobilized enzyme mixture, the pre-purified enzymes were immobilized using LX-1000 EP epoxy resin (Xi'an Lanxiao Company) in a ratio of 2:2:2:2:3 activity units. The basic immobilization method was as follows: 10,000 units of the enzyme mixture, mixed at the above activity unit ratio, were dissolved in 2 L of 50 mM potassium phosphate solution, pH 8.0. Phenoxyacetic acid (final concentration: 60 mM) and 900 g of LX-1000 EP 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 use. The immobilized YLpLigase / YLOxidase / NspLDH / BfaPPK / YLAmid enzyme mixture exhibited 75% to 92% of the activity of the corresponding liquid enzymes.

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

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

[0092] Example 1: Preparation of Cono Peptide Main Chain by Liquid Phase Synthesis Using Tripeptide Fragments as Raw Materials

[0093] Synthesis reaction see Figure 3 .

[0094] (1) Steps for connecting resin:

[0095] Wang resin (16.7 g, 10 mmol) with a substitution degree of 0.60 mmol / g was added to the reactor, allowed to swell in DMF for 30 min, and then filtered. 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. Stirring was maintained at 0–5°C for 5 min before the mixture was added to the reactor. The reaction was allowed to proceed at room temperature for 16 h, filtered, and washed three times with 60 mL of DMF. The Fmoc residue was removed twice with a 20% piperidine / DMF solution (10 min + 10 min), filtered, and washed six times with DMF before filtration.

[0096] (2) Coupling and deprotection steps

[0097] Fmoc-Asp(OtBu)-His(Trt)-Ala-OH (17.2 g, 20 mmol), HOBt (2.97 g, 22 mmol), and DIEA (5.2 mL, 30 mmol) were weighed and placed in a 250 mL beaker. 80 mL of DMF was added and stirred at 0–5°C until completely dissolved. DIC (2.78 g, 22 mmol) was added and stirred at 0–5°C for 5 min before being poured into the reactor. The reaction was allowed to proceed at room temperature for 1.5 h. Completion of the reaction was confirmed by ninhydrin. The product was filtered and washed three times with 60 mL of DMF. Fmoc removal was performed twice with 20% piperidine / DMF (10 min + 10 min). The solvent was removed under reduced pressure, and the product was washed six times with DMF before removal of the solvent under reduced pressure.

[0098] Repeat the above coupling and deprotection steps to sequentially condense the protected 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. In particular, the coupling time for Fmoc-Asn(Trt)-Gly-Pro was 5 h. After all amino acids were coupled, methanol was added to shrink the resin, resulting in 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) Cracking step:

[0101] Prepare a cleavage 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 cleavage buffer at 0–5°C, and maintain the reaction at 0–5°C with stirring for 3 h. After completion of the reaction, filter the resin, wash the resin three times with DCM, and collect the filtrate. Remove the TFA by vacuum concentration at 40–45°C. Then, slowly add the distillation residue dropwise to a large amount of icy tertiary 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, and wash three times with icy tertiary ether to collect the filter cake.

[0102] The filter cake was vacuum dried at 25°C to constant weight, and 15.9 g of cone snail peptide backbone product was collected with a yield of 73.3%. Liquid HPLC test confirmed that it was the cone snail peptide backbone. Figure 4 Seal tightly and store at -20℃ for future use.

[0103] Example 2: Using the conopeptide backbone as raw material, liquid enzymes (YLOxidase, NspLDH) oxidized the conopeptide backbone to prepare an oxidized conopeptide backbone

[0104] Synthesis reaction see Figure 5 .

[0105] In 1 L of 25 mM pH 8.0 Tris. HCl solution, add 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM) and 1.3 g of NAD +(2 mM) and then adjust the pH value of the solution back to 8.0. Then, add 2000 U YLOxidase crude enzyme solution and 2000 U NspLDH crude enzyme solution at one time to start the reaction. After stirring gently at 30°C for 3 hours, add hydrochloric acid to terminate the reaction, precipitate and centrifuge to remove the protein, and then use ammonium sulfate salting out or organic solvent precipitation to preliminarily enrich the target peptide. Then, ion exchange chromatography (IEX) is used for further purification based on charge difference, and small molecule impurities are removed and the molecular weight is determined by gel filtration chromatography (GFC). Reverse phase high performance liquid chromatography (RP-HPLC) is then used to achieve high-resolution purification based on hydrophobicity difference. Finally, 20.7 grams of oxidized conoside peptide as a white solid is obtained by drying (final yield 91%). The purified product is detected by HPLC and mass spectrometry (MS), then freeze-dried and stored at -80°C or in a vacuum desiccator. The low temperature operation is performed throughout the process to ensure stability. The test results are shown in the table. Figure 6 (HPLC), Figure 7 (Mass spectrometry).

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

[0107] Synthesis reaction see Figure 8 .

[0108] To 1 L of 25 mM pH 8.0 Tris-HCl solution, 22.7 g of oxidized conopeptide 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) were added. The pH value of the solution was adjusted back to 8.0, and then 2000 U of YLpLigase crude enzyme solution and 2000 U of ATP were added at once. 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; hydrochloric acid was added after 4 hours of reaction to terminate the reaction, and the protein was removed by precipitation and centrifugation. Subsequently, ammonium sulfate salting out or organic solvent precipitation was used to preliminarily enrich the target peptide; then further purification was performed by ion exchange chromatography (IEX) based on charge differences, and small molecule impurities were removed and the molecular weight was determined by gel filtration chromatography (GFC); then reversed-phase high-performance liquid chromatography (RP-HPLC) was used to achieve high-resolution purification based on hydrophobicity differences; finally, 21.0 g of a white solid with a pyroglutamated conus peptide backbone was obtained by drying (final yield 89%); the solid was freeze-dried and stored at -80°C or in a vacuum desiccator, and low-temperature operation was used throughout the process to ensure stability.

[0109] Example 4: Preparation of Conopeptide Using Pyroglutaminated Conopeptide Main Chain as Raw Material and Liquid Enzyme (YLAmid) as Catalyst

[0110] Synthesis reaction see Figure 9 .

[0111] To 1 L of 25 mM Tris-HCl (pH 8.0), 23.8 g of pyroglutaminated cono peptide backbone (10 mM) and 1.1 mL of concentrated ammonia (15 mM) were added. The pH of the solution was adjusted back to 8.0, and then 3000 U of YLAmid crude enzyme solution was added in one portion to initiate the reaction. The reaction was gently stirred at 30°C for 2 hours, and the reaction was terminated. The enzyme was precipitated by acid and centrifuged (~10,000 rpm, 10 minutes) to remove the precipitate. The target peptide was then initially enriched by ammonium sulfate precipitation or organic solvent precipitation. Further purification was performed by ion exchange chromatography (IEX) based on charge differences, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. Reverse-phase high-performance liquid chromatography (RP-HPLC) was then used to achieve high-resolution purification based on hydrophobicity differences. Finally, 20.4 g of cono peptide as a white solid was obtained by drying (final yield 86%). The purified product was confirmed by HPLC and mass spectrometry (MS). Figure 10 、 Figure 11 Freeze-dried and stored at -80°C or in a vacuum desiccator, the entire process is kept at low temperature to ensure stability.

[0112] Example 5: Preparation of conopeptide by one-step conversion using liquid enzymes (YLOxidase, NspLDH, YLpLigase, BfaPPK, YLAmid) using the conopeptide backbone as raw material

[0113] Synthesis reaction see Figure 12 .

[0114] In 1 L of 25 mM pH 8.0 Tris-HCl solution, 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), 1.3 g of NAD +(2 mM), 1.5 g pyroglutamic acid (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) were added to the flask to start the reaction. The reaction was stirred gently at 30°C. During the reaction, the pH of the reaction system was maintained between 7.0 and 9.0 with acid and alkali. The reaction was terminated after 6 hours, and the enzymes in the reaction solution were precipitated by acid and the reaction solution was quickly centrifuged (~10,000 μg / mL). rpm, 10 minutes) to remove enzyme precipitates, followed by initial enrichment of the target peptide using ammonium sulfate precipitation or organic solvent precipitation. Further purification was performed by ion exchange chromatography (IEX) based on charge differences, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. Reverse-phase high-performance liquid chromatography (RP-HPLC) was then used to achieve high-resolution purification based on hydrophobicity differences. Finally, drying yielded 19.4 g of cono peptide as a white solid (final yield 82%). The purified product was tested for purity by HPLC and mass spectrometry (MS), then lyophilized and stored at -80°C or in a vacuum desiccator, with low temperatures maintained throughout the process to ensure stability.

[0115] Example 6: Conopeptide backbone, NAD + , ATP, etc. as raw materials, and the immobilized mixed enzyme was used to convert conopeptide into

[0116] Synthesis reaction see Figure 13 .

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

[0118] In 1 L of 25 mM pH 8.0 Tris-HCl solution, 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), 1.3 g of NAD +The reaction was initiated by adding 10,000 U of the immobilized enzyme mix at once, followed by the addition of 10 mM glutamic acid (2 mM), 1.5 g of pyroglutamic acid (12 mM), 3.1 g of sodium hexametaphosphate (5 mM), 0.6 g of ATP (1 mM), 1.0 g of magnesium chloride hexahydrate (5 mM), and 1.1 mL of concentrated aqueous ammonia (15 mM). The pH of the solution was then adjusted back to 8.0, and the reaction was initiated by the addition of 10,000 U of the immobilized enzyme mix. The reaction was gently stirred at 35°C, and the pH was maintained between 7.0 and 8.5 throughout the reaction. After 8 hours, the reaction was complete, and the immobilized enzyme mix was collected by filtration (the enzyme mix retained 88% of its initial activity after six uses). The target peptide was initially enriched by ammonium sulfate salting out or organic solvent precipitation. Further purification was performed by ion exchange chromatography (IEX) based on charge differences, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. Reverse-phase high-performance liquid chromatography (RP-HPLC) was then used to achieve high-resolution purification based on hydrophobicity differences. Finally, 20.6 g of cono peptide was obtained as a white solid (final yield 87%) by drying. The purified product is tested for purity by HPLC and mass spectrometry (MS), then freeze-dried and stored at -80°C or in a vacuum desiccator. The entire process is operated at low temperature to ensure stability.

[0119] Comparative Example: Using the main chain of cono peptide as raw material, liquid enzymes (WTYLOxidase, WTNspLDH, WTYLpLigase, WTBfaPPK, WTYLAmid) were used for one-time conversion to prepare cono peptide

[0120] Synthesis reaction see Figure 14 .

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

[0122] In 1 L of 25 mM pH 8.0 Tris-HCl solution, 22.7 g of conopeptide backbone (10 mM), 2.9 g of pyruvate (33 mM), 1.3 g of NAD +(2 mM), 1.5 g pyroglutamic acid (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) were added to adjust the pH value of the solution back to 8.0, and 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 with acid and alkali during the reaction; the reaction was terminated after 6 hours, and the enzymes in the reaction solution were precipitated by acid and quickly centrifuged (~10000 rpm for 10 minutes) to remove enzyme precipitates. The target peptide was then initially enriched using ammonium sulfate precipitation or organic solvent precipitation. Further purification was performed using ion exchange chromatography (IEX) based on charge differences, followed by gel filtration chromatography (GFC) to remove small molecule impurities and determine the molecular weight. Reverse-phase high-performance liquid chromatography (RP-HPLC) was then used to achieve high-resolution purification based on hydrophobicity differences. Finally, 4.1 g of cono peptide was dried to obtain a white solid (final yield of 17%). The purified product was tested for purity by HPLC and mass spectrometry (MS), then lyophilized and stored at -80°C or in a vacuum desiccator. Low temperatures were maintained throughout the process to ensure stability.

[0123] The above is only a preferred embodiment 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 this application.

Claims

1. An enzyme mutant, characterized in that for: Pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1; or a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2; or an amino acid sequence of SEQ ID NO: 3; or Lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4; or An ATP regenerating enzyme having an amino acid sequence of SEQ ID NO:

5.

2. An enzyme combination, characterized in that The enzymes include a pyroglutamate ligase with an amino acid sequence of SEQ ID NO: 1, a sulfhydryl oxidase with an amino acid sequence of SEQ ID NO: 2, an aminase with an amino acid sequence of SEQ ID NO: 3, a lactate dehydrogenase with an amino acid sequence of SEQ ID NO: 4, and an ATP regenerator with an amino acid sequence of SEQ ID NO:

5.

3. An immobilized enzyme, characterized in that Comprising the enzyme mutant according to claim 1 or the enzyme combination according to claim 2.

4. The method for preparing an immobilized enzyme according to claim 3, wherein: include: mixing a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1, a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2, an aminase having an amino acid sequence of SEQ ID NO: 3, a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4, and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5 to obtain a mixed enzyme; The mixed enzyme is dissolved in a potassium phosphate solution, and then mixed with phenoxyacetic acid and epoxy resin, stirred, and filtered to obtain an immobilized enzyme; The pyroglutamate ligase, the sulfhydryl oxidase, the lactate dehydrogenase, the ATP regenerating enzyme, and the aminase are mixed in an enzyme activity unit ratio of 2:2:2:2:3, and the enzyme activity unit 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 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.

5. Use of the enzyme mutant according to claim 1, the enzyme combination according to claim 2, the immobilized enzyme according to claim 3, or the immobilized enzyme prepared by the preparation method according to claim 4 in the preparation of conopeptides.

6. A method for preparing conopeptide, characterized in that: include: Buffer, conopeptide backbone, pyruvate and NAD + Mixing, adjusting the pH to 7.0-9.0, and then mixing with a sulfhydryl oxidase having an amino acid sequence of SEQ ID NO: 2 and a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 4 to obtain a mixture 1, stirring, mixing with an acid, and separating and purifying to obtain an oxidized conopeptide backbone; Mixing a buffer, an oxidized conopeptide backbone, pyroglutamic acid, sodium hexametaphosphate, magnesium chloride, and ATP, adjusting the pH to 7.0-9.0, and then mixing with a pyroglutamate ligase having an amino acid sequence of SEQ ID NO: 1 and an ATP regenerating enzyme having an amino acid sequence of SEQ ID NO: 5 to obtain a mixture 2, stirring, maintaining the pH at 7.5-9.0, mixing with an acid, and separating and purifying to obtain a pyroglutamated conopeptide backbone; The buffer solution, the pyroglutamylated conopeptide backbone and concentrated ammonia were mixed to obtain a mixture 3, the pH of which was adjusted to 7.0-9.0, and the mixture was then mixed with an ammonia enzyme having an amino acid sequence of SEQ ID NO: 3, stirred, mixed with an acid, and separated and purified to obtain the conopeptide.

7. The preparation method according to claim 6, wherein The buffer is 1 L 25 mM pH 8.0 Tris-HCl; The content of the conopeptide backbone in the mixture 1 is 10 mM; The content of pyruvic acid in the mixture 1 is 33 mM; The NAD + The content in the mixture 1 is 2 mM; The content of the oxidized conopeptide backbone in the mixture 2 is 10 mM; The content of pyroglutamic acid in the mixture 2 is 12 mM; The content of sodium hexametaphosphate in the mixture 2 is 5 mM; The content of magnesium chloride in the mixture 2 is 5 mM; The content of ATP in the mixture 2 is 1 mM; The content of the pyroglutaminated conopeptide backbone in the mixture 3 is 10 mM; The content of the concentrated ammonia solution in the mixture 3 is 15 mM; The stirring temperature is 30°C; The acid is hydrochloric acid; The purification and separation comprises the steps of precipitation, centrifugation, ammonium sulfate salting out or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high performance liquid chromatography; The contents of the sulfhydryl oxidase and the lactate dehydrogenase in the mixture 1 are respectively 2000 U; The contents of the pyroglutamate ligase and the ATP regenerating enzyme in the mixture 2 are 2000 U respectively; The concentration of the aminotransferase was 3000 U.

8. A method for preparing conopeptide, characterized in that: include: Buffer, conopeptide backbone, pyruvate, NAD + , pyroglutamic acid, sodium hexametaphosphate, ATP, magnesium chloride, and concentrated ammonia water are mixed, the pH value is adjusted to 7.0-9.0, and then mixed with the immobilized enzyme to obtain a mixture, stirred, and the pH is maintained at 7.0-8.5, and separated and purified to obtain cono peptide; The immobilized enzyme is the immobilized enzyme according to claim 3 or the immobilized enzyme prepared by the preparation method according to claim 4.

9. The preparation method according to claim 8, wherein The buffer is 1 L 25 mM pH 8.0 Tris-HCl; The content of the conopeptide backbone in the mixture is 10 mM; The content of pyruvic acid in the mixture is 33 mM; The NAD + The content in the mixture was 2 mM; The content of pyroglutamic acid in the mixture is 12 mM; The content of sodium hexametaphosphate in the mixture is 5 mM; The content of magnesium chloride in the mixture is 5 mM; The content of ATP in the mixture is 1 mM; The content of the concentrated ammonia solution in the mixture is 15 mM; The stirring temperature is 35°C; The purification and separation comprises the steps of precipitation, centrifugation, ammonium sulfate salting out or organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, and reversed-phase high performance liquid chromatography.

10. The preparation method according to any one of claims 6 to 9, characterized in that: The cone peptide backbone was synthesized by a liquid phase synthesis method 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)-Cys(Trt)-OH.

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