Mutant of enzyme, enzyme composition and preparation method of Livagen

Abstract

The invention relates to the field of polypeptide synthesis, in particular to an enzyme mutant, an enzyme composition and a preparation method of Livagen. According to the invention, a plurality of mutant enzymes are used for combined catalytic conversion, common amino acids such as L-Asp, Ala, Lys, Glu and the like are used as initial raw materials, aspartic acid-alanine dipeptide and lysine-glutamic acid dipeptide are efficiently synthesized through liquid enzymes LaLigase and CspLigase respectively, and finally, the two dipeptides are used as substrates, and efficient synthesis of the target product Livagen is realized by means of catalytic coupling of the liquid enzyme LivaLigase. The enzyme method system has the advantages of mild reaction conditions, high conversion efficiency, few byproducts, easiness in industrial control and the like, so that the enzyme method system shows unique value in the aspect of green, efficient and large-scale production of Livagen, and an innovative path is provided for continuous preparation of high-purity Livagen in cosmetics and functional skin care products.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and particularly to enzyme mutants, enzyme compositions, and methods for preparing Livagen. Background Technology

[0002] Livagen (Lys-Glu-Asp-Ala) is a polypeptide complex extracted from natural plants, possessing both highly effective antioxidant and cell regeneration properties. Its mechanism of action involves activating the mitochondrial respiratory chain, enhancing ATP synthesis efficiency, and thus boosting cellular energy metabolism. Simultaneously, it regulates the Nrf2 / ARE pathway, promoting the expression of antioxidant enzyme systems, neutralizing free radicals, and delaying cell aging. In cosmetic applications, Livagen enhances keratinocyte activity, accelerates epidermal renewal, and improves rough and dull skin. Its antioxidant effect lasts for over 72 hours, providing both immediate brightening and long-lasting repair. Furthermore, this ingredient can enhance cell membrane stability, reduce oxidative stress caused by external stimuli, and improve skin's resilience and self-healing ability. Based on its unique synergistic effect of energy activation and cell protection, Livagen has significant application potential in anti-oxidative brightening skincare products, cell regeneration care, and damaged skin repair, especially suitable for high-energy skincare needs requiring both brightening and antioxidant effects.

[0003] Livagen, as a tetrapeptide, can be synthesized using two mainstream methods: solid-phase synthesis (SPPS) and liquid-phase synthesis (SPS). Although the patents / documents do not provide specific synthetic steps for Livagen, the described solid-phase synthesis techniques and improved methods are generally applicable to the synthesis of short peptides like Livagen. For example, the Fmoc-based solid-phase synthesis technique described in US20090171068A1 is suitable for its synthesis. It uses Wang or RinkAmide resin as the starting support, sequentially couples Fmoc-protected amino acids, and finally cleaves the peptide with TFA to remove the side-chain protecting groups. The target peptide is then purified by HPLC, offering advantages such as high efficiency, ease of automation, and suitability for industrial production. In contrast, the liquid-phase synthesis described in US20050004016A1 uses a Boc / Bz strategy, constructing and purifying dipeptide fragments stepwise before condensation. Although the operation is complex and the steps are cumbersome, it yields higher purity products suitable for applications requiring extremely high purity or large-scale production.

[0004] In existing technologies, 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; and enzymatic ligation methods are prone to producing sequence mismatch byproducts due to the limited substrate compatibility of enzymes. Summary of the Invention

[0005] In view of this, the present invention provides enzyme mutants, enzyme compositions, and methods for preparing Livagen. The present invention utilizes the combined catalytic transformation of multiple mutant enzymes, using common amino acids such as L-Asp, Ala, Lys, and Glu as starting materials, to efficiently synthesize aspartic acid-alanine dipeptide and lysine-glutamic acid dipeptide via liquid enzymes LaLigase and CspLigase, respectively. Finally, using these two dipeptides as substrates, the target product Livagen is efficiently synthesized through catalytic coupling with the liquid enzyme LivaLigase. Because this enzymatic system has advantages such as mild reaction conditions, high conversion efficiency, few byproducts, and ease of industrial control, it exhibits unique value in the green, efficient, and large-scale production of Livagen, providing an innovative pathway for the continuous preparation of high-purity Livagen in cosmetics and functional skincare products.

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

[0007] In a first aspect, the present invention provides mutants of enzymes, said mutants including one or more of lysine dipeptide ligase mutants, alanine dipeptide ligase mutants, liwagandide synthase mutants and / or ATP regenerator mutants.

[0008] (I) The lysine dipeptide ligase is derived from the actinomycete Conexibacter sp., Uniprot ID: A0AAE4CZ37;

[0009] The mutation sites of the mutant lysine dipeptide ligase include: V121, R122N, L126A, Q130D, I228V, R295H and / or S305T.

[0010] (II) The propionyl dipeptide ligase is derived from Bacillus alkalisoli (Uniprot ID: A0A917D3M4);

[0011] The mutation sites of the mutant of the acetyl-partate dipeptide ligase include: D31E, M180F, C182T, H183K and / or K255Q;

[0012] (III) The levapeptide synthase is derived from the actinomycete Streptomyces resistomycificus (Uniprot ID: A0A0L8KRM4);

[0013] The mutation sites of the mutants of the liwagen peptide synthase include: S47V, R83A, E92T, A115S, A116C, M124H, T144D, V240I, T241N, H251E, I358L and / or P430T.

[0014] (IV) The ATP regeneration enzyme is derived from Pedobacter nyackensis (UniprotID: A0A1W2DTD1);

[0015] The mutation sites of the mutant ATP regenerator include: G87V, R118H, H136N, Y139M and / or E237D.

[0016] In some specific embodiments of the present invention, the mutant of the lysine dipeptide ligase has:

[0017] (I) The amino acid sequence as shown in SEQ ID No. 1;

[0018] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0019] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II);

[0020] The plurality includes at least two integers;

[0021] and / or

[0022] The mutant of the acetyl-partate dipeptide ligase has:

[0023] (I) The amino acid sequence as shown in SEQ ID No. 2;

[0024] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0025] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II);

[0026] The plurality includes at least two integers;

[0027] and / or

[0028] The mutant of the leivagen peptide synthase has:

[0029] (I) The amino acid sequence as shown in SEQ ID No. 3;

[0030] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0031] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II);

[0032] The plurality includes at least two integers;

[0033] and / or

[0034] The mutant of the ATP regenerating enzyme has:

[0035] (I) The amino acid sequence shown in SEQ ID No. 4;

[0036] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0037] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II);

[0038] The plurality includes at least two integers.

[0039] Secondly, the present invention also provides a nucleic acid molecule encoding a mutant of the enzyme.

[0040] In some specific embodiments of the present invention, the nucleic acid molecule includes one or more of the following: a mutant of the lysine dipeptide ligase, a mutant of the lysine dipeptide ligase, a mutant of the liwagen peptide synthase, and / or a mutant of the ATP regenerator.

[0041] The nucleic acid molecule encoding the mutant of the glutathione dipeptide ligase has:

[0042] (1) A nucleotide sequence as shown in SEQ ID NO.5; or

[0043] (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0044] (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2);

[0045] and / or

[0046] The nucleic acid molecule encoding the mutant of the allypeptide ligase has:

[0047] (1) A nucleotide sequence as shown in SEQ ID NO. 6; or

[0048] (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0049] (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2);

[0050] and / or

[0051] The nucleic acid molecule encoding the mutant of the leivagenin synthase has:

[0052] (1) A nucleotide sequence as shown in SEQ ID NO.7; or

[0053] (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0054] (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2);

[0055] and / or

[0056] The nucleic acid molecule encoding the mutant of the ATP regenerating enzyme has:

[0057] (1) A nucleotide sequence as shown in SEQ ID NO. 8; or

[0058] (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0059] (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2).

[0060] Thirdly, the present invention also provides an enzyme composition comprising a mutant of the enzyme;

[0061] Preferably, the enzyme composition includes a mutant of the lysine dipeptide ligase, a mutant of the acetyl-partate dipeptide ligase, a mutant of the livagin peptide synthase, and a mutant of the ATP regenerator.

[0062] Preferably, the enzyme composition comprises liquid enzymes and / or immobilized enzymes.

[0063] Fourthly, the present invention also provides a fusion protein of a mutant of the enzyme.

[0064] Fifthly, the present invention also provides a nucleic acid molecule encoding the enzyme composition or the fusion protein described herein.

[0065] In a sixth aspect, the present invention also provides an expression element, including the aforementioned nucleic acid molecule.

[0066] In a seventh aspect, the present invention also provides an expression vector, including the aforementioned expression element.

[0067] In an eighth aspect, the present invention also provides a host, said host:

[0068] (A) A mutant expressing the enzyme; and / or

[0069] (B) Expressing the enzyme composition described herein; and / or

[0070] (C) Expressing the fusion protein; and / or

[0071] (D) Transformation or transfection of the expressed element; and / or

[0072] (E) Transformation or transfection with the expression vector described above;

[0073] Preferably, the host includes microorganisms and / or cells.

[0074] In a ninth aspect, the present invention also provides the following applications in the preparation of Livagen:

[0075] (i) A mutant of the enzyme; and / or

[0076] (ii) the aforementioned nucleic acid molecules; and / or

[0077] (iii) the enzyme composition described above; and / or

[0078] (iv) the fusion protein; and / or

[0079] (v) the aforementioned expressive element; and / or

[0080] (vi) the aforementioned expression carrier; and / or

[0081] (vii) The host mentioned above.

[0082] In a tenth aspect, the present invention also provides a method for preparing Livagen, comprising:

[0083] (a) Using L-Asp, Ala, Lys, or Glu as substrates, mix with a mutant of the enzyme, the enzyme composition, or the fusion protein to obtain Livagen; or

[0084] (b) Using L-Asp, Ala, Lys, and Glu as substrates, express the aforementioned nucleic acid molecules, and mix the obtained protein product with the substrates to obtain Livagen; or

[0085] (c) Using L-Asp, Ala, Lys, or Glu as substrates, transform or transfect the expression element or the expression vector described above, and mix the obtained protein product with the substrate to obtain Livagen; or

[0086] (d) Using L-Asp, Ala, Lys, and Glu as substrates, the host is cultured, and the obtained protein products are mixed with amino acids to obtain Livagen.

[0087] In some specific embodiments of the present invention, the preparation method includes the following steps:

[0088] Using L-Asp and Ala as raw materials, aspartic acid-alanine dipeptide was prepared by catalysis of the mutant of the alanine dipeptide ligase and the mutant of the ATP regenerating enzyme.

[0089] Using Lys and Glu as raw materials, and catalyzed by mutants of the lysine-glutamic acid dipeptide ligase and mutants of the ATP regenerating enzyme, a lysine-glutamic acid dipeptide was prepared.

[0090] Livagen was prepared using the aspartic acid-alanine dipeptide and the lysine-glutamic acid dipeptide as raw materials, catalyzed by a mutant of the Livagen peptide synthase and the ATP regenerator.

[0091] In some specific embodiments of the present invention, the preparation method is as follows: using L-Asp, Ala, Lys and Glu as raw materials, and catalyzing the enzyme composition or fusion protein, Livagen is prepared.

[0092] This patent is the first to report the use of multiple mutant enzymes in catalytic transformation, using common amino acids such as L-Asp, Ala, Lys, and Glu as starting materials. Aspartic acid-alanine dipeptide and lysine-glutamic acid dipeptide are efficiently synthesized via liquid enzymes LaLigase and CspLigase, respectively. Finally, using these two dipeptides as substrates, the target product Livagen is efficiently synthesized through catalytic coupling with the liquid enzyme LivaLigase. Because this enzymatic system has advantages such as mild reaction conditions, high conversion efficiency, few byproducts, and ease of industrial control, it demonstrates unique value in the green, efficient, and large-scale production of Livagen, providing an innovative pathway for the continuous preparation of high-purity Livagen in cosmetics and functional skincare products. Attached Figure Description

[0093] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0094] Figure 1 The SDS-PAGE information of the enzymes used is shown; where M: Marker; 1: CspLigase, 2: LaLigase, 3: LivaLigase, 4: PnPPK;

[0095] Figure 2 Thin-layer chromatography (TLC) was used to detect the reaction products of Lys-Glu and Asp-Ala. The developing system was n-butanol / acetic acid / water in a volume ratio of 4:1:1. The colorimetric reagent was 0.4% ninhydrin-acetone solution. In the left figure (A), 1 shows glutamic acid, 2 shows lysine, 4 shows the Lys-Glu dipeptide reaction solution, and 3 shows the mixing point of the raw materials and reaction solution. In the right figure (B), 1 shows aspartic acid, 3 shows the Asp-Ala dipeptide reaction solution, and 2 shows the mixing point of the raw materials and reaction solution.

[0096] Figure 3 This is an HPLC detection of Livagen tetrapeptide; mobile phase A: 0.1% H3PO4 aqueous solution; mobile phase B: acetonitrile; detection wavelength: 205 nm; where A represents the reaction solution of Example 3, B represents the reaction solution of Example 4, C represents the reaction solution of Example 5, and D represents the comparative example.

[0097] Figure 4 Livagen 1 H-NMR, D2O as solvent, Varian 600 M NMR;

[0098] Figure 5 Livagen 13 C-NMR with D2O as solvent, Varian 600 M NMR. Detailed Implementation

[0099] This invention discloses enzyme mutants, enzyme compositions, and methods for preparing Livagen. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0100] The multi-step enzymatic route for preparing Livagen in this invention:

[0101]

[0102] Table 1

[0103]

[0104] Information regarding the enzymes used above:

[0105] Lysine dipeptide ligase (CspLigase): Derived from the actinomycete Conexibacter sp. (UniprotID: A0AAE4CZ37), this natural enzyme (WTCspLigase) has certain activities for L-lysine and glutamate. After site-directed modification (CspLigase), its activity is significantly improved. The specific mutation sites are: V121, R122N, L126A, Q130D, I228V, R295H, and S305T.

[0106] Aspartate dipeptide ligase (LaLigase): Derived from Bacillus alkalisoli (Uniprot ID: A0A917D3M4), this natural enzyme (WTLaLigase) has certain activities for aspartic acid and alanine. After systematic modification, the expression and activity of (LaLigase) have been improved. The specific mutation sites are: D31E, M180F, C182T, H183K, and K255Q.

[0107] LivaLigase: Derived from the actinomycete *Streptomyces resistomycificus*, Uniprot ID: A0A0L8KRM4, this natural enzyme (WTLivaligase) exhibits weak activity in the linkage of allyndeptide and lysine dipeptide, and its expression and stability are not ideal. Through comprehensive modification of this enzyme (Livaligase), all of the above performance aspects are significantly improved. Specific mutation sites are: S47V, R83A, E92T, A115S, A116C, M124H, T144D, V240I, T241N, H251E, I358L, and P430T.

[0108] ATP regenerase (PnPPK): Pedobacter nyackensis (Uniprot ID: A0A1W2DTD1). The natural enzyme (WTPnPPK) has high activity for ATP regeneration, but its expression level and stability are not ideal. The performance of the mutant enzyme (PnPPK) after modification is significantly improved. The specific mutation sites are: G87V, R118H, H136N, Y139M, and E237D.

[0109] Table 2

[0110]

[0111] Table 3

[0112]

[0113]

[0114] Table 4: Information on the properties of the enzymes used in this patent.

[0115]

[0116] This invention innovatively employs an enzymatic synthesis strategy, using common amino acids such as L-Asp, Ala, Lys, and Glu as starting materials. Aspartic acid-alanine dipeptide and lysine-glutamic acid dipeptide are efficiently synthesized using liquid enzymes LaLigase and CspLigase, respectively. Finally, using these two dipeptides as substrates, a directed-evolution-modified liquid enzyme (LivaLigase) is used for catalytic coupling, achieving efficient synthesis of Livagen. This method avoids the complex protecting group operations of traditional synthesis, significantly reduces the generation of byproducts, and utilizes the high substrate specificity of the enzyme system to achieve precise peptide chain splicing, greatly improving synthesis efficiency and product purity. This process provides an efficient and environmentally friendly new path for the continuous and green production of cosmetic-grade high-purity Livagen, effectively solving the core challenges of traditional methods in terms of efficiency, cost, and environmental friendliness.

[0117] The preparation of Livagen begins with L-Asp and Ala as starting materials, and involves the efficient synthesis of an aspartic-alanine dipeptide catalyzed by a liquid enzyme (LaLigase, PnPPK). Then, using Lys and Glu as starting materials, a lysine-glutamic acid dipeptide is synthesized using a liquid enzyme (CspLigase, PnPPK). Finally, using these two dipeptides as substrates, Livagen is precisely synthesized through coupling catalysis by liquid enzymes (LivaLigase, PnPPK) that have undergone multiple redirection evolution modifications. The enzyme system described above, through directed evolution technology, significantly improves its catalytic efficiency, substrate compatibility, and thermal stability, enabling the entire synthesis process to proceed efficiently under mild conditions. This method avoids the complex protecting group operations and the use of toxic solvents in traditional multi-step chemical synthesis, significantly reducing byproduct formation and improving product purity and synthesis efficiency.

[0118] The raw materials and reagents used in the enzyme mutants, enzyme compositions, and Livagen preparation methods provided by this invention are all commercially available.

[0119] The present invention will be further illustrated below with reference to the embodiments:

[0120] Preparation Example 1: Fermentation Production of Enzymes:

[0121] The enzymes used in this patent 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 is synthesized by a gene company (Anhui General Biotechnology). Then, it is subcloned into the pET28a plasmid through the NdeI / XhoI restriction site. The plasmid is then transformed into E. coli (BL21) (Qingke Biotechnology) cells for plate culture. Finally, single colonies are selected for liquid step-by-step scale-up culture. The following is the basic procedure for cell step-by-step scale-up culture: First, single colonies on the plate are transferred into 5 ml of LB medium containing 50 μM kanamycin (37°C).o C) Culture the cells. Once they reach the logarithmic growth phase, seed them into 250 ml of LB medium containing the same antibiotic, and then transfer them to a 5 L fermenter for further culture. When the cell OD reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) 30. o C induces protein expression for 5 hours, then centrifuges (4000 rpm, 15 min) to collect 20-30 g of wet cells. To verify enzyme expression, a small amount of cells is first mixed with Tris-HCl buffer (50 mM, pH 8.0), followed by cell lysis using a freeze-thaw method. After high-speed centrifugation, the supernatant is run on an SDS-PAGE protein gel (sodium dodecyl sulfonate-polyacrylamide gel) to confirm soluble protein expression (e.g., ...). Figure 1 (As shown); confirm the correct remaining cells and mix them with buffer (10g of wet cells mixed with approximately 200ml of the above buffer). Then, perform high-pressure cell disruption and high-speed centrifugation (16000 rpm, 10 min) to remove the cell wall. The resulting enzyme-containing supernatant can be used directly for subsequent applications (the enzyme activity is 100-1000 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). The LB medium consists of: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0122] Preparation Example 2: Mixed Immobilization of Enzymes:

[0123] Ammonium sulfate solid was added incrementally to the crude enzyme solutions of glutathione dipeptide ligase (CspLigase), lyvaglutide synthase (LaLigase), livaglutide synthase (LivaLigase), and ATP regenerator (PnPPK) collected in Preparation Example 1 until enzyme precipitation (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000 rpm, 15 min) and slowly dissolved in 25 mM pH 8.0 Tris buffer. Finally, the enzymes were 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 CspLigase, LaLigase, LivaLigase, and PnPPK. In the immobilized mixed enzyme, the above pre-purified enzymes were immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at a ratio of 2:2:2:3 activity units. The basic immobilization method is as follows: 9000U of mixed enzymes, prepared according to the above activity unit ratio, are dissolved in 2L of 50 mM pH 8.0 potassium phosphate solution. Then, 60 mM phenoxyacetic acid and 900g of LX-1000 EP epoxy resin are added to the buffer solution. After stirring at room temperature for 8 hours, the immobilized enzyme is filtered out. Finally, it is washed three times each with water and 25 mM pH 8.0 phosphate buffer, and then dried at low temperature for later use. The CspLigase / LaLigase / LivaLigase / PnPPK immobilized mixed enzyme has 70-95% of the activity of the corresponding liquid enzyme.

[0124] Example 1: Preparation of aspartic acid-alanine dipeptide (Asp-Ala) using L-Asp and Ala as raw materials and liquid enzyme (LaLigase, PnPPK)

[0125]

[0126] The pH of a 1 L solution of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) was adjusted to 8.0 by adding 4.0 g L-Asp (30 mM), 3.2 g L-Ala (36 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 6.7 g sodium hexametaphosphate (11 mM), and 0.6 g ATP (1 mM). Then, 2000 U of crude LaLigase enzyme solution and 1000 U of crude PnPPK enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 38°C. oC. Stir gently, and maintain the pH of the reaction system between 7.5 and 8.5 using acid and base during the reaction. After 2 hours of reaction, add hydrochloric acid to terminate the reaction, precipitate, centrifuge to remove protein, then adjust the solution to pH 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing the product is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 5.7 g of Asp-Ala dipeptide as a white solid (e.g. Figure 2 As shown in the figure, the final yield was 93%.

[0127] Example 2: Preparation of Lysine-Glutamic Acid Dipeptide (Lys-Glu) using Lysine and Glutamic Acid as raw materials via liquid enzyme (CspLigase, PnPPK)

[0128]

[0129] The pH of a 1 L solution of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) was adjusted to 8.0 by adding 4.4 g L-Lys (30 mM), 3.2 g L-Glu (36 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 6.7 g sodium hexametaphosphate (11 mM), and 0.6 g ATP (1 mM). Then, 2000 U of crude CspLigase enzyme solution and 1000 U of crude PnPPK enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 38°C. o C. Stir gently, and maintain the pH of the reaction system between 7.5 and 8.5 using acid and base during the reaction. After 2 hours, add hydrochloric acid to terminate the reaction, precipitate, centrifuge to remove protein, then adjust the solution to pH 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing the product is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 7.5 g of Lys-Glu dipeptide white solid (as shown in the image). Figure 2 As shown in the figure, the final yield was 91%.

[0130] Example 3: Preparation of Livagen (Lys-Glu-Asp-Ala) using aspartic-alanine dipeptide and lysine-glutamic acid dipeptide as raw materials via liquid enzymes (LivaLigase, PnPPK)

[0131]

[0132] The pH of a 1 L solution of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) was adjusted to 8.0 by adding 5.5 g Lys-Glu (30 mM), 4.9 g Asp-Ala (36 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 6.7 g sodium hexametaphosphate (11 mM), and 0.6 g ATP (1 mM). Then, 2000 U LivaLigase crude enzyme solution and 1000 U PnPPK crude enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 38°C. o C. Stir gently, and maintain the pH of the reaction system between 7.5 and 8.5 using acid and base during the reaction. After 4 hours of reaction, add hydrochloric acid to terminate the reaction, precipitate, centrifuge to remove protein, then adjust the solution to pH 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing the product is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 8.0 g of Lys-Glu-Asp-Ala tetrapeptide white solid (as shown in the image). Figures 3-5 As shown in the figure, the final yield was 87%.

[0133] Example 4: Preparation of Livagen from lysine, glutamic acid, aspartic acid, and alanine using liquid enzymes (CspLigase, LaLigase, LivaLigase, PnPPK) in a single conversion.

[0134]

[0135] The pH of the solution was adjusted to 8.0 by adding 4.4 g L-Lys (30 mM), 5.3 g L-Glu (36 mM), 4.8 g L-Asp (36 mM), 3.2 g L-Ala (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22 g sodium hexametaphosphate (36 mM), and 0.6 g ATP (1 mM) to 1 L of 25 mM pH 8.0 Tris. Then, 2000 U LivaLigase crude enzyme solution, 2000 U LivaLigase crude enzyme solution, 2000 U LivaLigase crude enzyme solution, and 3000 U PnPPK crude enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 38°C. oC. Stir gently, and maintain the pH of the reaction system between 7.5 and 8.5 using acid and base during the reaction. After 6 hours of reaction, add hydrochloric acid to terminate the reaction, precipitate, centrifuge to remove protein, then adjust the solution to pH 7.0 and remove phosphate-containing impurities using D201 anion exchange resin. The effluent containing the product is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 7.0 g of Lys-Glu-Asp-Ala tetrapeptide white solid (as shown in the image). Figures 3-5 As shown in the figure, the final yield was 85%.

[0136] Example 5: Preparation of Livagen via a single-step conversion of an immobilized mixed enzyme using lysine, glutamic acid, aspartic acid, and alanine as raw materials.

[0137]

[0138] The reaction is similar to that in Example 4 above, but an immobilized enzyme is used, so it can be recycled multiple times.

[0139] The pH of the solution was adjusted to 8.0 by adding 4.4 g L-Lys (30 mM), 5.3 g L-Glu (36 mM), 4.8 g L-Asp (36 mM), 3.2 g L-Ala (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22 g sodium hexametaphosphate (36 mM), and 0.6 g ATP (1 mM) to 1 L of 25 mM pH 8.0 Tris. o C. With gentle stirring, the pH of the reaction system was maintained between 7.5 and 8.5 using acid and base during the reaction. After 8 hours, hydrochloric acid was added to terminate the reaction. The protein was precipitated and centrifuged to remove it. The solution was then adjusted to pH 7.0 and phosphate-containing impurities were removed using D201 anion exchange resin. The effluent containing the product was then purified and collected using D101 nonpolar resin. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 7.2 g of Lys-Glu-Asp-Ala tetrapeptide white solid (as shown in the image). Figures 3-5 As shown in the figure, the final yield was 87%.

[0140] Comparative Example: Livagen was prepared by one-step conversion of lysine, glutamic acid, aspartic acid, and alanine using liquid enzymes (WTCspLigase, WTLaLigase, WTLivaLigase, WTPnPPK).

[0141]

[0142] Similar to Example 4 above, each enzyme was replaced with the natural enzyme WT.

[0143] The pH of the solution was adjusted to 8.0 by adding 4.4 g L-Lys (30 mM), 5.3 g L-Glu (36 mM), 4.8 g L-Asp (36 mM), 3.2 g L-Ala (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22 g sodium hexametaphosphate (36 mM), and 0.6 g ATP (1 mM) to 1 L of 25 mM pH 8.0 Tris. Then, 4000 U WTLivaLigase crude enzyme solution, 4000 U WTLivaLigase crude enzyme solution, 4000 U WTLivaLigase crude enzyme solution, and 5000 U WTPnPPK crude enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 38°C. o C. Stir gently, and maintain the pH of the reaction system between 7.5 and 8.5 using acid and base during the reaction. After 10 hours of reaction, add hydrochloric acid to terminate the reaction, precipitate, centrifuge to remove protein, then adjust the solution to pH 7.0 and use D201 anion exchange resin to remove phosphate impurities. The effluent containing the product is then purified and collected using D101 nonpolar resin. Finally, the crude product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:H2O=1:1, V:V) to obtain 0.94 g of Lys-Glu-Asp-Ala tetrapeptide white solid (as shown in the image). Figures 3-5 As shown in the figure, the final yield was 11%.

[0144] 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 the present invention.

Claims

1. A mutant of an enzyme, characterized in that, The mutants of the enzyme include one or more of the following: lysine dipeptide ligase mutants, alanine dipeptide ligase mutants, liwagandide synthase mutants, and / or ATP regenerator mutants. (I) The lysine dipeptide ligase is derived from the actinomycete Conexibacter sp., Uniprot ID: A0AAE4CZ37; The mutation sites of the mutant lysine dipeptide ligase include: V121, R122N, L126A, Q130D, I228V, R295H and / or S305T. (II) The propionyl dipeptide ligase is derived from Bacillus alkalisoli (UniprotID: A0A917D3M4); The mutation sites of the mutant of the acetyl-partate dipeptide ligase include: D31E, M180F, C182T, H183K and / or K255Q; (III) The levapeptide synthase is derived from the actinomycete Streptomyces resistomycificus (Uniprot ID: A0A0L8KRM4); The mutation sites of the mutants of the liwagen peptide synthase include: S47V, R83A, E92T, A115S, A116C, M124H, T144D, V240I, T241N, H251E, I358L and / or P430T. (IV) The ATP regenerating enzyme is derived from Pedobacter nyackensis (Uniprot ID: A0A1W2DTD1); The mutation sites of the mutant ATP regenerator include: G87V, R118H, H136N, Y139M and / or E237D.

2. The mutant of the enzyme as described in claim 1, characterized in that, The mutant of the lysine dipeptide ligase has: (I) The amino acid sequence as shown in SEQ ID No. 1; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II); The plurality includes at least two integers; and / or The mutant of the acetyl-partate dipeptide ligase has: (I) The amino acid sequence as shown in SEQ ID No. 2; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II); The plurality includes at least two integers; and / or The mutant of the leivagen peptide synthase has: (I) The amino acid sequence as shown in SEQ ID No. 3; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II); The plurality includes at least two integers; and / or The mutant of the ATP regenerating enzyme has: (I) The amino acid sequence shown in SEQ ID No. 4; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in any one of (I) or (II); The plurality includes at least two integers.

3. A nucleic acid molecule encoding a mutant of the enzyme as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleic acid molecule includes one or more of the following: a mutant of the lysine dipeptide ligase, a mutant of the lysine dipeptide ligase, a mutant of the liwagen peptide synthase, and / or a mutant of the ATP regenerator. The nucleic acid molecule encoding the mutant of the glutathione dipeptide ligase has: (1) A nucleotide sequence as shown in SEQ ID NO.5; or (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2); and / or The nucleic acid molecule encoding the mutant of the allypeptide ligase has: (1) A nucleotide sequence as shown in SEQ ID NO. 6; or (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2); and / or The nucleic acid molecule encoding the mutant of the leivagenin synthase has: (1) A nucleotide sequence as shown in SEQ ID NO.7; or (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2); and / or The nucleic acid molecule encoding the mutant of the ATP regenerating enzyme has: (1) A nucleotide sequence as shown in SEQ ID NO. 8; or (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (1) or (2).

5. An enzyme composition, characterized in that, Including mutants of the enzyme as described in claim 1 or 2; Preferably, the enzyme composition includes a mutant of the lysine dipeptide ligase, a mutant of the acetyl-partate dipeptide ligase, a mutant of the livagin peptide synthase, and a mutant of the ATP regenerator. Preferably, the enzyme composition comprises liquid enzymes and / or immobilized enzymes.

6. A fusion protein of a mutant of the enzyme as described in claim 1 or 2.

7. A nucleic acid molecule, characterized in that, Encodes the enzyme composition as described in claim 5 or the fusion protein as described in claim 6.

8. An expression element, characterized in that, Includes nucleic acid molecules as described in claims 3, 4 or 7.

9. An expression carrier, characterized in that, Includes the expression element as described in claim 8.

10. The host, characterized in that, The host: (A) A mutant expressing the enzyme as described in claim 1 or 2; and / or (B) Expressing the enzyme composition as described in claim 5; and / or (C) Expressing the fusion protein as described in claim 6; and / or (D) Transformation or transfection with the expression element as described in claim 8; and / or (E) Transformation or transfection with the expression vector as described in claim 9; Preferably, the host includes microorganisms and / or cells.

11. Any of the following applications in the preparation of Livagen: (i) A mutant of the enzyme as described in claim 1 or 2; and / or (ii) The nucleic acid molecule as described in claim 3, 4 or 7; and / or (iii) The enzyme composition as described in claim 5; and / or (iv) The fusion protein as described in claim 6; and / or (v) The expression element as described in claim 8; and / or (vi) The expression vector as described in claim 9; and / or (vii) The host as described in claim 10.

12. A method for preparing Livagen, characterized in that, include: (a) Using L-Asp, Ala, Lys, or Glu as substrates, mix with a mutant of the enzyme as described in claim 1 or 2, the enzyme composition as described in claim 5, or the fusion protein as described in claim 6 to obtain Livagen; or (b) Using L-Asp, Ala, Lys, or Glu as substrates, express the nucleic acid molecule as described in claim 3, 4, or 7, and mix the obtained protein product with the substrate to obtain Livagen; or (c) Using L-Asp, Ala, Lys, or Glu as substrates, transform or transfect the expression element as described in claim 8 or the expression vector as described in claim 9, and mix the obtained protein product with the substrate to obtain Livagen; or (d) Using L-Asp, Ala, Lys, and Glu as substrates, the host as described in claim 10 is cultured, and the obtained protein product is mixed with amino acids to obtain Livagen.

13. The preparation method according to claim 12, characterized in that, Includes the following steps: Using L-Asp and Ala as raw materials, aspartic acid-alanine dipeptide was prepared by catalysis of the mutant of the alanine dipeptide ligase and the mutant of the ATP regenerating enzyme. Using Lys and Glu as raw materials, and catalyzed by mutants of the lysine-glutamic acid dipeptide ligase and mutants of the ATP regenerating enzyme, a lysine-glutamic acid dipeptide was prepared. Livagen was prepared using the aspartic acid-alanine dipeptide and the lysine-glutamic acid dipeptide as raw materials, catalyzed by a mutant of the Livagen peptide synthase and the ATP regenerator.

14. The preparation method according to claim 12, characterized in that, Livagen was prepared by using L-Asp, Ala, Lys and Glu as raw materials and catalyzing the enzyme composition or fusion protein.

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