Compositions of enzyme mutants and their application in the preparation of bis(lauramide-glutamine) lysine salt

Abstract

This invention relates to the field of enzyme engineering technology, specifically to compositions of enzyme mutants and their application in the preparation of bis(lauramide-glutamine)lysine salt. This invention provides a process for the all-enzymatic synthesis of bis(lauramide-glutamine)lysine salt or its sodium salt. This process fully utilizes the high efficiency and substrate specificity of enzymatic catalysis, avoids the complex chemical reactions and protecting group operations of traditional chemical synthesis, significantly reduces the generation of byproducts, and simultaneously achieves site-specific modification and precise assembly of the target product. This technology provides an efficient and green solution for the continuous production of cosmetic-grade high-purity bis(lauramide-glutamine)lysine salt or its sodium salt, and has significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and more specifically to compositions of enzyme mutants and their application in the preparation of bis(lauramide-glutamine) lysine salt. Background Technology

[0002] Sodium di(lauramide-glutamine)lysine is a di(lauramide-glutamine)lysine salt, a dicationic amino acid surfactant synthesized from natural amino acids (glutamic acid and lysine) through laurate esterification. It possesses both excellent emulsifying stability and gentle skin affinity. Its molecular structure incorporates both hydrophilic and hydrophobic groups, resulting in good compatibility and stability in oil-in-water and water-in-oil emulsion systems. As a novel bioactive emulsifier, it not only provides gentle cleansing, low irritation, and good skin compatibility, but also gently removes excess oil and dirt by regulating the skin's surface charge balance, while maintaining the hydration of the stratum corneum and preventing barrier damage caused by excessive degreasing. Its dicationic structure effectively neutralizes anionic impurities, enhancing cleansing power and foam stability. Furthermore, this ingredient mimics the structure of natural phospholipid membranes, enhancing the affinity between the emulsion and the skin, and promoting the penetration and absorption of active ingredients; its tripeptide structural units can also gently regulate the skin's microecology, strengthen the skin barrier function, and alleviate sensitivity issues such as dryness and redness. In formulation applications, sodium di(lauramide-glutamine)lysine not only serves as a highly effective emulsifier but also synergizes with anti-aging, soothing, and moisturizing active ingredients to enhance the overall efficacy and user experience of the product. Its low allergenicity, high biocompatibility, and environmentally friendly biodegradability make it an ideal choice for high-end skincare, sensitive skin care, and green formulation development.

[0003] Currently, the main method for preparing sodium di(lauramide-glutamine)lysine is chemical synthesis. As an amphiphilic amino acid derivative, it is typically synthesized through multi-step chemical reactions, with three main synthetic processes. The first, based on patent WO2004 / 020394, involves reacting N-lauroyl glutamic anhydride with lysine in water or an aqueous / organic solvent to generate the target compound, which is then converted to its sodium salt. The second is a two-step method: first, L-glutamic acid is acylated with lauric acid under alkaline conditions to generate dilauroyl glutamic acid, which then condenses with lysine in the presence of a dehydrating agent to form an amide bond, and finally neutralized to form the sodium salt. The third is a one-pot method, where lauric acid, L-glutamic acid, and L-lysine are directly condensed under catalysis and heating conditions. The reaction is driven by removing the water generated during the reaction, and the final product is obtained through neutralization. However, these traditional chemical synthesis methods typically involve complex multi-step reactions and protecting group operations, limiting their application in high-purity cosmetic-grade products. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a composition of enzyme mutants and its application in the preparation of bis(lauramide-glutamine)lysine salt. The present invention provides a technical solution for preparing bis(lauramide-glutamine)lysine salt or its sodium salt by using L-lysine salt, L-glutamate salt and lauric acid as raw materials, and a synergistic catalytic system of esterase and ligase modified by directed evolution to achieve efficient and selective coupling of lauroylglutamate and lysine.

[0005] This invention provides a composition of enzyme mutants, the composition comprising glutathione dipeptide ligase, glutathione tripeptide ligase, lauroyl glutathione tripeptide synthase, and ATP regenerator, wherein:

[0006] The glutathione dipeptide ligase is derived from Photobacterium piscicola, and the mutation sites of the glutathione dipeptide ligase include at least one of D75E, A177S, Y186T, E315H and N424D.

[0007] The glutathione-glutathione tripeptide ligase is derived from Enterobacillus tribolii, and the mutation sites of the glutathione-glutathione tripeptide ligase include at least one of D77Q, M79I, R84H, N186K, S201V, I204L, P302Q, G322L and E325N.

[0008] The lauroyl glucoside tripeptide synthase is derived from Mycobacteroides abscessus, and the mutation sites of the lauroyl glucoside tripeptide synthase include at least one of L18A, E91D, E93K, L100F, V172S, L173G, R194E, V198I, T288S, I423W, and T424H.

[0009] The ATP regenerator is derived from Clostridium thailandense, and the mutation sites of the ATP regenerator include at least one of G70N, D72N, I75M, V134T, and R211E.

[0010] In some embodiments, the composition comprises a mutant of glutathione dipeptide ligase, a mutant of glutathione tripeptide ligase, a mutant of lauroyl glutathione tripeptide synthase, and a mutant of ATP regenerator, wherein:

[0011] The mutant of the glutathione dipeptide ligase has the amino acid sequence shown in SEQ ID NO:1, the mutant of the glutathione tripeptide ligase has the amino acid sequence shown in SEQ ID NO:2, the mutant of the lauroyl glutathione tripeptide synthase has the amino acid sequence shown in SEQ ID NO:3, and the mutant of the ATP regenerator has the amino acid sequence shown in SEQ ID NO:4.

[0012] The present invention provides an immobilized enzyme, comprising a composition of the enzyme mutant described above.

[0013] This invention provides nucleic acid molecules encoding at least one of the following (1) and / or (2):

[0014] (1) The composition of the enzyme mutant;

[0015] (2) The immobilized enzyme.

[0016] In some embodiments, the nucleic acid molecule includes a mutant encoding a glutathione dipeptide ligase, a mutant encoding a glutathione tripeptide ligase, a mutant encoding a lauroyl glutathione tripeptide synthase, and a mutant encoding an ATP regenerator, wherein:

[0017] The nucleic acid molecule of the mutant encoding glutathione dipeptide ligase has the nucleotide sequence shown in SEQ ID NO:5, the nucleic acid molecule of the mutant encoding glutathione tripeptide ligase has the nucleotide sequence shown in SEQ ID NO:6, the nucleic acid molecule of the mutant encoding lauroyl glutathione tripeptide synthase has the nucleotide sequence shown in SEQ ID NO:7, and the nucleic acid molecule of the mutant encoding ATP regenerator has the nucleotide sequence shown in SEQ ID NO:8.

[0018] This invention provides an expression vector or host cell, wherein the expression vector comprises the aforementioned nucleic acid molecule;

[0019] The host cell is transfected or transformed with the expression vector.

[0020] This invention provides the use of at least one of the following ① to ④ in the preparation of bis(lauramide-glutamine) lysine salt:

[0021] ① The composition of the enzyme mutant;

[0022] ② The immobilized enzyme;

[0023] ③ The aforementioned nucleic acid molecules;

[0024] ④ The expression vector or host cell.

[0025] In some specific embodiments, the di(lauramide-glutamine)lysine salt is sodium di(lauramide-glutamine)lysine.

[0026] This invention provides a method for preparing bis(lauramide-glutamine)lysine salt, characterized in that bis(lauramide-glutamine)lysine salt is prepared by using L-lysine salt, L-glutamate salt and lauric acid as raw materials and undergoing at least one of the following conversions (I to IV);

[0027] I. The composition of the enzyme mutant described above;

[0028] II. The immobilized enzyme;

[0029] III. The aforementioned nucleic acid molecules;

[0030] IV. The expression vector or host cell.

[0031] In some embodiments, the preparation method includes stepwise preparation or one-time preparation:

[0032] The stepwise preparation includes:

[0033] Using L-lysine salt and L-glutamate salt as raw materials, glutamate-lysine dipeptide is generated by catalysis of the mutant of the glutamate-lysine dipeptide ligase and the mutant of the ATP regenerating enzyme.

[0034] The glutamic acid-lysine dipeptide is catalyzed by the mutant of the glutamic-lysine-glutamic tripeptide ligase and the mutant of the ATP regenerating enzyme to generate glutamic acid-lysine-glutamic acid tripeptide.

[0035] After the glutamic acid-lysine-glutamic acid tripeptide is catalyzed and deproteinized by the mutant of the lauroyl glutaryl tripeptide synthase and the mutant of the ATP regenerator, the resulting solution generates the di(lauramide glutamine) lysine salt under the action of sodium hydroxide.

[0036] The one-time preparation involves taking L-lysine salt, L-glutamate salt, lauric acid, a mutant of glutathione dipeptide ligase, a mutant of glutathione tripeptide ligase, a mutant of lauroyl glutathione tripeptide synthase, and a mutant of ATP regenerator, mixing them, removing proteins, and then reacting the resulting solution with sodium hydroxide to generate the di(lauramide glutamine) lysine salt.

[0037] In some embodiments, characterized in that,

[0038] The ratio of the mutants of L-lysine, L-glutamate, lauric acid, glutathione dipeptide ligase, glutathione tripeptide ligase, lauroyl glutathione tripeptide synthase and ATP regenerator is (20~50mM): (50~80mM): (20~50mM): (500~1500U): (500~1500U): (500~1500U): (3000~5000U).

[0039] In some specific embodiments, the ratio of L-lysine salt, L-glutamate salt, lauric acid, mutant glutathione dipeptide ligase, mutant glutathione tripeptide ligase, mutant lauroyl glutathione tripeptide synthase, and mutant ATP regenerator is 30mM:66mM:36mM:1000U:1000U:1000U:4500U.

[0040] In some specific embodiments, the di(lauramide-glutamine)lysine salt is sodium di(lauramide-glutamine)lysine, the L-lysine salt is sodium L-lysine, and the L-glutamate salt is sodium L-glutamate.

[0041] This invention provides a process for the all-enzymatic synthesis of di(lauramide-glutamine)-lysine salt or its sodium salt. Starting with L-glutamate or its sodium salt (L-Glu) and L-lysine or its sodium salt (L-Lys), a glutamate-lysine dipeptide is efficiently prepared using the liquid enzyme PpLigase. Based on this, the dipeptide is linked to glutamate using the liquid enzyme EtLigase to generate a glutamate-lysine-glutamate tripeptide. Finally, using the tripeptide and lauric acid as raw materials, di(lauramide-glutamine)-lysine salt or its sodium salt is precisely synthesized using the liquid enzyme LauEKEMaLigase. This process fully utilizes the high efficiency and substrate specificity of enzymatic catalysis, avoiding the complex chemical reactions and protecting group operations of traditional chemical synthesis, significantly reducing the generation of byproducts, and achieving site-specific modification and precise assembly of the target product. This technology provides an efficient and green solution for the continuous production of cosmetic-grade high-purity di(lauramide-glutamine)-lysine salt or its sodium salt, and has significant industrial application value. Attached Figure Description

[0042] Figure 1 This invention illustrates the synthetic route for the total enzymatic synthesis of sodium di(lauramide-glutamine)lysine.

[0043] Figure 2 The image shows an SDS-PAGE gel image of the enzyme prepared in this invention, where M: protein standard marker, 1 is CtPPK, 2 is LauEKEMaSyn, 3 is EtLigase, and 4 is PpLigase.

[0044] Figure 3 The synthesis reaction formula of Example 2 is shown;

[0045] Figure 4 The synthesis reaction formula of Example 3 is shown;

[0046] Figure 5 The synthesis reaction formula of Example 4 is shown;

[0047] Figure 6 The synthesis reaction formula of Example 5 is shown;

[0048] Figure 7 The HPLC detection results of sodium bis(lauramide-glutamine)lysine prepared in Example 5 are shown.

[0049] Figure 8 The sodium bis(lauramide-glutamine)lysine prepared in Example 5 is shown. 1 H-NMR detection results;

[0050] Figure 9 The synthesis reaction formula of Example 6 is shown;

[0051] Figure 10 The synthetic reaction formula is shown in the comparative example. Detailed Implementation

[0052] This invention provides compositions of enzyme mutants and their application in the preparation of sodium bis(lauramide-glutamine)lysine. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0053] In existing technologies, the traditional esterification method for synthesizing bis(lauramide-glutamine) lysine salt or its sodium salt requires high temperature and pressure, relies on strong acid and strong base catalysts, has harsh reaction conditions, high energy consumption, and produces many byproducts, making it difficult to meet the requirements of green production. Stepwise condensation methods require multiple purifications of intermediates, resulting in long process flows, high costs, and unstable yields. Solid-phase synthesis methods are limited by resin loading and elution conditions, easily introducing impurities and affecting the purity of the final product. This patent innovatively adopts a total enzymatic synthesis strategy (preparation route as follows) Figure 1As shown in the figure, a highly efficient and selective coupling of lauroyl glutamate and lysine is achieved through a synergistic catalytic system of esterase and ligase modified by directed evolution. The reaction is carried out at ambient temperature and pressure, requiring no toxic solvents or catalysts, significantly reducing energy consumption and pollution. Simultaneously, the multi-enzyme cascade reaction design enables a continuous and high-throughput reaction pathway, greatly improving product yield and purity. This method not only simplifies the synthesis process and reduces production costs, but also provides a feasible route for the green, efficient, and large-scale production of cosmetic-grade di(lauramide-glutamine) lysine salt or its sodium salt, effectively solving the core problems of harsh reaction conditions, severe pollution, and high costs in traditional processes.

[0054] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0055] Example 1: Preparation of Four Liquid Enzymes

[0056] This embodiment provides four liquid enzymes, including glutathione dipeptide ligase (PpLigase), glutathione tripeptide ligase (EtLigase), lauroyl glutathione tripeptide synthase (LauEKEMaSyn), and ATP regenerase (CtPPK).

[0057] 1. Specific information about the enzyme:

[0058] Glutathione dipeptide ligase (PpLigase): Derived from Photobacterium piscicola (Uniprot ID: A0A1T5I2V4), this natural enzyme (WTPpLigase) has certain activities for L-glutamic acid and L-lysine. After site-directed modification, its activity and stability were improved. The specific mutation sites are: D75E, A177S, Y186T, E315H, and N424D.

[0059] The amino acid sequence of PpLigase:

[0060] MTFSERLQQVAKNPEALTQLGRGLERESLRITEDLQVSSLPHPTALGSALTNKWITTDFAESLLEFITPVSRDVEDLLAQLDDIHKFSLSHMAGERLWPMSMPCFVGDQDEIELAQYGSSNTGQMKTLYREGLKRRYGSVMQIISGVHFNFSFPDTFWDSLFGDQTEQQRQDSVSDSYFGLIRNYTRFGWLIPYLFGSSPALCGSFIQNEALKASFEKVGKGTYFLENATALRLSDLGYTNHAQSSLKIGFNSIDQYLTGLNSAIHTPSEEFSELGVMVDGKRQQLNSNVLQIENELYAPIRPKRVAKSGEKPSHALKRAGVEYIEVRSLDVNPFSPIGIDEDQVRFLDLFLIWATLTPSPDMTDCELACWRENWNKVVVDGRNPELLLKIGCHGEELLLKAWGRRVFAELEQVAITLDGLYGDDKYQQTHQRLLTWIEDPQLTFSAKLLDQIKTYQGIGPLGDYYATAHAKQLAQQAFRFYDQATFEQEVAASVIKQHQIEQSDTLSFDEFLADYFADVDVEIPMLTK (SEQ ID NO:1)

[0061] Nucleotide sequence of PpLigase:

[0062]

[0063] EtLigase, derived from Enterobacillus tribolii (Uniprot ID: A0A370Q8C0), is a natural enzyme with very weak activity for L-glutamate and L-glutamate-lysine dipeptides. Through systematic modification, its expression and activity have been improved. Specific mutation sites are: D77Q, M79I, R84H, N186K, S201V, I204L, P302Q, G322L, and E325N.

[0064] The amino acid sequence of EtLigase:

[0065] MIPDVSKALTWLEAHPDALNGIRRGIERETLRVTPDGHLAQTGHPAVLGKAFTHPWITTDFAETLLEFITPVDASIQHILAFLHDIHRYVARNLGNERMWPMSMPCFIGKEEDIVLAQYGTSNQGRFKTLY REGLKNRYGALMQTISGVHYNFSLPIEFWQAWAGVTDAESGKEKISAGYFRLIRKYYRFGWVIPYLFGAVPALCSSFLNGRETNLPFERSGKGMLYLPYATSLRLSDLGYTNKSQSNLGITFNDLDGYVTAL KKAIHTPSPEFARLGVNVDGHYRQLNANVLQIENELYAQIRPKRVTKGSESPSDALLRLGINYIEVRLSLDINPFTAIGVNAEQSRFLDLFLIWCVLADAPEMSSEELMCTRKNWNRVILEGRKPGQTIGIG CDSAREPLDKVGKSLFADLYRVAEVLDSINGNEQYQRVCTKLVTFFDDVSQTYSARVLEAMKSQGIGGFGLSLAEGYREALCHEPFEVLTPAMLDEQQKKSVEKQAMLEAQDTISFEEYLALHAGR (SEQID NO:2)

[0066] The nucleotide sequence of EtLigase:

[0067]

[0068] Lauroyl glutathione synthase (LauEKEMaSyn): Derived from Mycobacterium abscessus Uniprot ID: B1MCS0, this enzyme has three domains, and this transformation utilizes the AMP-binding domain (1~540 aa). The native enzyme (WTLauEKEMaSyn) has a weak synthetic ability, and its catalytic efficiency and stability are not ideal. Through comprehensive modification of this enzyme (LauEKEMaSyn), both aspects of its performance are significantly improved. The specific mutation sites are: L18A, E91D, E93K, L100F, V172S, L173G, R194E, V198I, T288S, I423W, and T424H.

[0069] The amino acid sequence of LauEKEMaSyn:

[0070] (SEQ ID NO:3)

[0071] The nucleotide sequence of LauEKEMaSyn:

[0072]

[0073] ATP regenerator (CtPPK): Clostridium thailandense, Uniprot ID: A0A949X149. The natural enzyme (WTCtPPK) has good ATP regeneration activity, but its expression level and stability are not ideal. The mutant enzyme (CtPPK) has significantly improved performance after modification. The specific mutation sites are: G70N, D72N, I75M, V134T, and R211E.

[0074] The amino acid sequence of CtPPK:

[0075] MNVSEFRVTNKSKFKLNDIKTSYTGDFNSKEDAQKHLIKNIEQMSEIQSKLYAQGKYGILIIFQAMDTGNKNSAMKHVMSGLNPQGTKVYSFKEPSAEELSHDYLWKAHKHIPERGQIGIFNRSYYEELLVVRTHNLIKNQRIPE EFITDSIWKKRFEQIKNFEKYLYENGIIPIKIFLHISKEEQKKRLLERINDKTKNWKFSESDIKEESHWDKYQQFYEEAIRETSSKSIPWFVVPADKKWFARLVISQIIIDKLEELKLEYPTLSKEQYDGLEECRKKLIEE (SEQ ID NO:4)

[0076] The nucleotide sequence of CtPPK:

[0077] atgaacgtgagcgaatttcgcgtgaccaacaaaagcaaatttaaactgaacgatattaaaaccagctataccggcgattttaacagcaaagaagatgcgcagaaacatctgattaaaaacattgaacagatgagcgaaattcagagcaaactgtatgcgcagggcaaatatggcattctgattatttttcaggcgatggataccggcaacaaaaacagcgcgatgaaacatgtgatgagcggcctgaacccgcagggcaccaaagtgtatagctttaaagaaccgagcgcggaagaactgagccatgattatctgtggaaagcgcataaacatattccggaacgcggccagattggcatttttaaccgcagctattatgaagaactgctggtggtgcgcacccataacctgattaaaaaccagcgcattccggaagaatttattaccgatagcatttggaaaaaacgctttgaacagattaaaaactttgaaaaatatctgtatgaaaacggcattattccgattaaaatttttctgcatattagcaaagaagaacagaaaaaacgcctgctggaacgcattaacgataaaaccaaaaactggaaatttagcgaaagcgatattaaagaagaaagccattgggataaatatcagcagttttatgaagaagcgattcgcgaaaccagcagcaaaagcattccgtggtttgtggtgccggcggataaaaaatggtttgcgcgcctggtgattagccagattattattgataaactggaagaactgaaactggaatatccgaccctgagcaaagaacagtatgatggcctggaagaatgccgcaaaaaactgattgaagaataa(SEQ ID NO:8)

[0078] 2. Fermentation production of enzyme:

[0079] 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 25, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). o C-induced protein expression was performed for 10 hours, followed by centrifugation (4000 rpm, 15 min) to collect 20-35 g of wet cells. To verify enzyme expression, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were lysed using a freeze-thaw method. After high-speed centrifugation, the supernatant was run on an SDS-PAGE protein gel (sodium dodecyl sulfonate-polyacrylamide gel) to confirm soluble protein expression. The remaining cells, after confirmation, were mixed with buffer (10 g of wet cells mixed with approximately 200 ml of the above buffer), and then subjected to high-pressure cell lysis and high-speed centrifugation (16000 rpm, 10 min) to remove the cell wall. The resulting enzyme-containing supernatant was used directly for subsequent applications (the liquid enzyme activity is 250-1200 U / ml, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or further purified and immobilized for use (in solid enzyme reactions). LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol. The obtained crude enzyme solutions containing glutathione dipeptide ligase (PpLigase), glutathione tripeptide ligase (EtLigase), lauroyl glutathione tripeptide synthase (LauEKEMaSyn), and ATP regenerator (CtPPK) were analyzed by SDS-PAGE gel chromatography. The results are as follows: Figure 2 As shown.

[0080] 3. Mixed immobilization of enzymes:

[0081] Ammonium sulfate solid was added incrementally to the collected crude enzyme solutions of glutathione dipeptide ligase (PpLigase), glutathione tripeptide ligase (EtLigase), lauroyl glutathione tripeptide synthase (LauEKEMaSyn), and ATP regenerator (CtPPK) 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 PpLigase, EtLigase, LauEKEMaSyn, and CtPPK. In the immobilized mixed enzyme, the pre-purified enzymes were immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at a ratio of 1:1:1:4.5 activity units. The basic immobilization method is as follows: 8000U 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 PpLigase / EtLigase / LauEKEMaSyn / CtPPK immobilized mixed enzyme has 75-92% of the activity of the corresponding liquid enzyme.

[0082] Example 2: Preparation of glutamate-lysine dipeptide (L-Glu-Lys) using L-Glu and L-Lys as raw materials via liquid enzyme (PpLigase, CtPPK)

[0083] Synthesis reaction formula as follows Figure 3 As shown. In 1 L of 25 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution, 4.4 g L-Lys sodium salt (30 mM), 5.3 g L-Glu sodium salt (36 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 6.7 g sodium hexametaphosphate (11 mM), and 0.6 g ATP (1 mM) were added to adjust the pH to 8.0. Then, 1000 U of PpLigase crude enzyme solution and 1500 U of CtPPK 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 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 7.6 g of L-Glu-Lys dipeptide white solid (final yield 92%).

[0084] Example 3: Glutamic acid-lysine-glutamic acid tripeptide (Glu-Lys-Glu) was prepared from glutamic acid-lysine dipeptide and glutamic acid using liquid enzymes (EtLigase, CtPPK).

[0085] Synthesis reaction formula as follows Figure 4 As shown. 6.9 g L-Glu-Lys (25 mM), 4.4 g L-Glu (30 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 6.1 g sodium hexametaphosphate (10 mM), and 0.6 g ATP (1 mM) were added to 1 L of 25 mM pH 8.0 Tris. The pH of the solution was adjusted to 8.0. Then, 1000 U U of crude Ligase enzyme solution and 1500 U of crude CtPPK 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 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 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 9.0 g of Glu-Lys-Glu tripeptide white solid (final yield 89%).

[0086] Example 4: Preparation of sodium di(lauramide-glutamine)lysine using glutamic acid-lysine-glutamic acid tripeptide (Glu-Lys-Glu) and lauric acid as raw materials via liquid enzymes (LauEKEMaLigase, CtPPK)

[0087] Synthesis reaction formula as follows Figure 5As shown. In 1 L of 25 mM pH 8.0 Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution, 8.1 g Glu-Lys-Glu (20 mM), 4.8 g lauric acid (24 mM), 3.0 g magnesium chloride hexahydrate (15 mM), 4.9 g sodium hexametaphosphate (8 mM), and 0.6 g ATP (1 mM) were added to adjust the pH to 8.0. Then, 1000 U U LauEKEMaLigase crude enzyme solution and 1500 U CtPPK crude enzyme solution were added simultaneously to initiate the reaction. The reaction was carried out at 30°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 of reaction, slowly add hydrochloric acid to the reaction solution to adjust the pH to 4 and precipitate solid. Centrifuge to remove protein precipitate, collect the target solid in a water / acetonitrile system (ethanol:H2O=1:1, V:V) and slurry. After filtering again, add it to purified water and stir evenly. Then, slowly add 1 mol / L sodium hydroxide aqueous solution to the solution until the pH reaches 7.6. After freeze-drying, 13.6 g of sodium bis(lauroyl glutamine) lysine sodium white solid (final yield 89%) is obtained.

[0088] Example 5: Sodium di(lauramide-glutamine)lysine was prepared by a one-step conversion of lysine, glutamic acid, and lauric acid using liquid enzymes (PpLigase, EtLigase, LauEKEMaSyn, CtPPK).

[0089] Synthesis reaction formula as follows Figure 6 As shown. In 1 L of 25 mM pH 8.0 Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution, 4.4 g L-Lys sodium salt (30 mM), 9.7 g L-Glu sodium salt (66 mM), 7.2 g lauric acid (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22.0 g sodium hexametaphosphate (36 mM), and 1.7 g ATP (3 mM) were added to adjust the pH to 8.0. Then, 1000 U PpLigase crude enzyme solution, 1000 U EtLigase crude enzyme solution, 1000 U U LauEKEMaLigase crude enzyme solution, and 4500 U CtPPK crude enzyme solution were added simultaneously to initiate the reaction. 30 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, slowly add hydrochloric acid to the reaction solution to adjust the pH to 4 and precipitate solid. Centrifuge to remove protein precipitate, collect the target solid in a water / acetonitrile system (ethanol:H2O=1:1, V:V) and slurry. After filtration again, add it to purified water and stir evenly. Then, slowly add 1 mol / L sodium hydroxide aqueous solution to the solution until the pH reaches 7.6. After freeze-drying, 18.8 g of sodium bis(lauroyl glutamine) lysine sodium white solid (final yield 81%) is obtained.

[0090] The products were verified using 1H NMR spectroscopy and HPLC, respectively, and the results are as follows: Figure 7 and Figure 8 As shown, the purity of sodium di(lauroyl glutamine) lysine in the final product is 97%.

[0091] Example 6: Sodium di(lauramide-glutamine)lysine was prepared by immobilized enzyme through a one-time conversion using lysine, glutamic acid, and lauric acid as raw materials.

[0092] The reaction is similar to that in Example 5 above, but an immobilized enzyme is used, so it can be recycled multiple times.

[0093] Synthesis reaction formula as follows Figure 9 As shown. In 1 L of 25 mM pH 8.0 Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution, 4.4 g L-Lys sodium salt (30 mM), 9.7 g L-Glu sodium salt (66 mM), 7.2 g lauric acid (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22.0 g sodium hexametaphosphate (36 mM), and 1.7 g ATP (3 mM) were added to adjust the pH to 8.0. Then, 8000 U of immobilized enzyme solution was added at once to initiate the reaction. 30 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 8 hours of reaction, slowly add hydrochloric acid to the reaction solution to adjust the pH to 4 and precipitate solid. Centrifuge to remove protein precipitate, collect the target solid in a water / acetonitrile system (ethanol:H2O=1:1, V:V) and slurry. After filtering again, add it to purified water and stir evenly. Then, slowly add 1 mol / L sodium hydroxide aqueous solution to the solution until the pH reaches 7.6. After freeze-drying, 19.6 g of sodium bis(lauroyl glutamine) lysine sodium white solid (final yield 85%) is obtained.

[0094] In the comparative example, sodium di(lauramide-glutamine)lysine was prepared by a one-step conversion using lysine, glutamic acid, and lauric acid as raw materials and liquid enzymes (WTPpLigase, WTEtLigase, WTLauEKEMaSyn, WTCtPPK).

[0095] Similar to Example 5 above, each enzyme was replaced with the natural enzyme WT.

[0096] Synthesis reaction formula as follows Figure 10 As shown. In 1 L of 25 mM pH 8.0 Tris(hydroxymethyl)aminomethane hydrochloride (Tris. HCl) solution, 4.4 g L-Lys sodium salt (30 mM), 9.7 g L-Glu sodium salt (66 mM), 7.2 g lauric acid (36 mM), 6.1 g magnesium chloride hexahydrate (30 mM), 22.0 g sodium hexametaphosphate (36 mM), and 1.7 g ATP (3 mM) were added to adjust the pH to 8.0. Then, 1000 U WTPpLigase crude enzyme solution, 1000 U WTEtLigase crude enzyme solution, 1000 U WTLauEKEMaLigase crude enzyme solution, and 4500 U WTCtPPK crude enzyme solution were added simultaneously to initiate the reaction. 30 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 6 hours of reaction, slowly add hydrochloric acid to the reaction solution to adjust the pH to 4 and precipitate solid. Centrifuge to remove the protein precipitate, collect the target solid in a water / acetonitrile system (ethanol:H2O=1:1, V:V) and slurry. After filtering again, add it to purified water and stir evenly. Then, slowly add 1 mol / L sodium hydroxide aqueous solution to the solution until the pH reaches 7.6. After freeze-drying, 2.0 g of sodium bis(lauroyl glutamine) lysine sodium white solid (final yield 9%) is obtained.

[0097] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition of enzyme mutants, characterized in that, The composition comprises a mutant of glutathione dipeptide ligase, a mutant of glutathione tripeptide ligase, a mutant of lauroyl glutathione tripeptide synthase, and a mutant of ATP regenerator, wherein: The amino acid sequence of the mutant glutathione dipeptide ligase is shown in SEQ ID NO:1, the amino acid sequence of the mutant glutathione tripeptide ligase is shown in SEQ ID NO:2, the amino acid sequence of the mutant lauroyl glutathione tripeptide synthase is shown in SEQ ID NO:3, and the amino acid sequence of the mutant ATP regenerator is shown in SEQ ID NO:

4.

2. An immobilized enzyme, characterized in that, A composition comprising the enzyme mutant of claim 1.

3. Nucleic acid molecules that encode at least one of the following (1) and / or (2): (1) The composition of the enzyme mutant according to claim 1; (2) The immobilized enzyme as described in claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that, This includes nucleic acid molecules encoding mutants of glutathione dipeptide ligase, mutants of glutathione tripeptide ligase, mutants of lauroyl glutathione tripeptide synthase, and mutants of ATP regenerator, wherein: The nucleotide sequence of the nucleic acid molecule encoding the mutant glucolysine dipeptide ligase is shown in SEQ ID NO:5, the nucleotide sequence of the nucleic acid molecule encoding the mutant glucolysine tripeptide ligase is shown in SEQ ID NO:6, the nucleotide sequence of the nucleic acid molecule encoding the mutant lauroyl glucolysine tripeptide synthase is shown in SEQ ID NO:7, and the nucleotide sequence of the nucleic acid molecule encoding the mutant ATP regenerator is shown in SEQ ID NO:

8.

5. An expression vector or host cell, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3 or 4; The host cell is transfected or transformed with the expression vector.

6. The following at least one of ① to ④ is used in the preparation of bis(lauramide-glutamine)-lysine salt: ① The composition of the enzyme mutant according to claim 1; ② The immobilized enzyme as described in claim 2; ③ The nucleic acid molecule as described in claim 3 or 4; ④ The expression vector or host cell as described in claim 5.

7. A method for preparing sodium bis(lauramide-glutamine)lysine, characterized in that, Using L-lysine salt, L-glutamate salt and lauric acid as raw materials, bis(lauramide-glutamine)lysine salt is prepared by at least one of the following conversions I to IV; I. The composition of the enzyme mutant according to claim 1; II. The immobilized enzyme as described in claim 2; III. The nucleic acid molecule as described in claim 3 or 4; IV. The expression vector or host cell as described in claim 5.

8. The preparation method according to claim 7, characterized in that, The preparation method includes: Using L-lysine salt and L-glutamate salt as raw materials, glutamate-lysine dipeptide is generated by catalysis of the mutant of the glutamate-lysine dipeptide ligase and the mutant of the ATP regenerating enzyme. The glutamic acid-lysine dipeptide is catalyzed by the mutant of the glutamic-lysine-glutamic tripeptide ligase and the mutant of the ATP regenerating enzyme to generate glutamic acid-lysine-glutamic acid tripeptide. After the glutamic acid-lysine-glutamic acid tripeptide is catalyzed and deproteinized by the mutant of the lauroyl glutaryl tripeptide synthase and the mutant of the ATP regenerator, the resulting solution generates the di(lauramide glutamine) lysine salt under the action of sodium hydroxide.

9. The preparation method according to claim 8, characterized in that, The ratio of the mutants of L-lysine, L-glutamate, lauric acid, glutathione dipeptide ligase, glutathione tripeptide ligase, lauroyl glutathione tripeptide synthase and ATP regenerator is (20~50mM): (50~80mM): (20~50mM): (500~1500U): (500~1500U): (500~1500U): (3000~5000U).

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