L-carnosine, preparation method thereof, and aminopeptidase

Abstract

The present invention belongs to the field of synthetic biology, and specifically relates to a kind of L-carnosine and its preparation method and aminopeptidase. The present invention is based on the theory of synthetic biology and develops a preparation method of L-carnosine based on homemade aminopeptidase and produces high-purity L-carnosine. The preparation method of L-carnosine of the present invention can achieve the recovery and reuse of L-histidine while obtaining high-purity L-carnosine, effectively reducing costs. In addition, the preparation method of the present invention has mild reaction conditions, a short synthetic route, is environmentally friendly, and the product has high optical purity.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology, and specifically relates to L-carnosine, a preparation method thereof, and aminopeptidase. Background Art

[0002] L-carnosine (N-β-Alanyl-L-histidine), also known as β-alanyl-L-histidine, molecular formula: C9H 14 N₄O₃, with a molecular weight of 226.23, is a crystalline dipeptide derived from the condensation of the amino acids β-alanine and L-histidine. First discovered in beef in 1900, it is widely found in the brain, muscle, and other tissues of mammals. It is a naturally active dipeptide also known as L-carnosine. L-carnosine exhibits antioxidant and anti-aging properties, and is effective in treating hypertension, heart disease, senile cataracts, ulcers, and other conditions. It also has anti-tumor and other biological activities. Adding carnosine to fresh meat during storage can enhance its flavor and extend its shelf life. L-carnosine has been shown to scavenge reactive oxygen species (ROS) and α-β-unsaturated aldehydes formed by the excessive oxidation of fatty acids in cell membranes during oxidative stress. Adding carnosine to cosmetics can delay skin aging. Compared to other antioxidants, L-carnosine offers advantages such as strong antioxidant capacity, a lack of toxic side effects, and multiple physiological activities, offering broad application prospects in medicine, healthcare, hygiene, and beauty. Currently, the exploration of new physiological activities and mechanisms of L-carnosine remains a hot topic.

[0003] Currently, L-carnosine production methods fall into two main categories: chemical synthesis and biosynthesis. Chemical synthesis requires complex protection and deprotection steps for the active groups of the substrates β-alanine and L-histidine. This leads to complex synthesis steps, harsh reaction conditions, low product yields, high pollution levels, and the presence of toxic reagent residues.

[0004] Biosynthesis offers advantages such as mild reaction conditions, short synthesis routes, environmental friendliness, and high product optical purity. Therefore, obtaining high-yield L-carnosine has always been a challenge, and developing an efficient enzyme capable of catalyzing the synthesis of carnosine is crucial. Summary of the Invention

[0005] In order to solve the technical problem of how to obtain high-yield L-carnosine in the above background technology, the present invention provides L-carnosine and a preparation method thereof and aminopeptidase.

[0006] The technical solution adopted in the present invention is as follows:

[0007] In a first aspect, the present invention provides an aminopeptidase for catalyzing the synthesis of L-carnosine. The amino acid sequence of the aminopeptidase is shown in SEQ ID NO: 1.

[0008] The beneficial effects of the present invention are: the aminopeptidase independently developed by the present invention has high catalytic efficiency and can obtain L-carnosine with high yield and purity, thus solving the enzyme problem in biosynthesis of L-carnosine.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, the nucleotide sequence of the aminopeptidase is shown in SEQ ID NO: 2.

[0011] A second aspect of the present invention provides a method for preparing the aminopeptidase as described above, comprising the steps of:

[0012] S1. Search and screen dipeptidases in the database;

[0013] S2. introducing the dipeptidase encoding gene described in step S1 into the recipient through plasmid construction and transformation, and finally screening the suitable template enzyme by detecting the enzymatic activity of the dipeptidase;

[0014] S3, performing mutation modification on the template enzyme in step S2, and then repeating step S2 to screen and obtain the aminopeptidase with the highest enzyme activity.

[0015] On the basis of the above technical solution, the present invention can also be improved as follows.

[0016] Furthermore, in step S2, the recipient is the strain Escherichia coli BL21.

[0017] The beneficial effect of the present invention is that, through the above steps, self-made aminopeptidase can be obtained, and subsequently, based on the aminopeptidase, effective biosynthesis of β-alanine and L-histidine can be achieved to obtain L-carnosine.

[0018] A third aspect of the present invention provides a method for preparing L-carnosine, comprising the steps of:

[0019] S1, inoculating the aminopeptidase according to any one of claims 1 to 3 and fermenting the resulting β-aminopeptidase as an auxiliary modification enzyme, using β-alanine and L-histidine as substrates to react and obtain L-carnosine;

[0020] S2, the L-carnosine in step S1 is decolorized and then filtered through an ultrafiltration membrane. The resulting ultrafiltration membrane clear solution is further purified by resin to obtain analytical solution 1 and analytical solution 2. The analytical solution 2 is then concentrated and crystallized after removing impurities through a nanofiltration membrane, and then dried after recrystallization and purification to obtain high-purity L-carnosine.

[0021] On the basis of the above technical solution, the present invention can also be improved as follows.

[0022] Furthermore, in step S2, the decolorization is performed using activated carbon.

[0023] Furthermore, in step S2, the analytical solution 1 is concentrated, crystallized, and then dried to obtain L-histidine.

[0024] Furthermore, in step S2, the resin is LKC60 resin.

[0025] Furthermore, in step S2, the nanofiltration membrane is an 80D membrane core.

[0026] The present invention has the beneficial effects of not only obtaining L-carnosine but also obtaining high-purity L-carnosine, and enabling the recovery and reuse of L-histidine, thereby effectively reducing costs. Furthermore, the process of the present invention has mild reaction conditions, a short synthesis route, is environmentally friendly, and produces a product with high optical purity.

[0027] A fourth aspect of the present invention provides L-carnosine, which is prepared using the above-mentioned method for preparing L-carnosine.

[0028] The beneficial effects of the present invention are that the L-carnosine obtained by the preparation method of the present invention has high purity, high preparation efficiency and high yield, and can provide high-quality L-carnosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a process flow chart of the present invention;

[0030] Figure 2 It is the schematic diagram of L-carnosine synthesis principle of the present invention;

[0031] Figure 3 It is a liquid phase detection spectrogram of L-carnosine of the present invention;

[0032] The information of the sequences involved in the present invention is described in the following table:

[0033] SEQ ID NO: describe sequence 1 Amino acid sequence of HCYJ-13 MSELSQLSPQPLWDIFAKICSIPHPSYHEEALAQHILTWAKEKNLHAERDQVGNILLRKPATKGMENRKPVALQAHLDMVPQKNNDTVHDFAKDPIQPYIAGEWVKARGTSLGADNGIGMA SALAVLADDSVEHGPLEVLLTMTEEAGMDDAFGLQPNWLQADILINTDSEEEGEIYMGCAGGIDFITTLPLQREAVPAGYQTLKLTLKGLKGGHSGAEIHVGLGNANKLLARFLFAHAAALN LRVLDLNGGTLRNAIPREASAVVAVPADKADALKALSQEFLAVLQNELSAKEKNITVLLEPTTSASLALSADSQQRFLALLNGTPNGVIRMSDAVKGVVETSLNVGVVTTSENEAEIICLI RSLIDSGKDYVVEMLTALGQLAGAKVAPKGGYPGWQPDADSPVMHLVRELYQDLFNKTPNIMVIHAGLECGLFKKPYPNMDMVSIGPTITGPHSPDEQVHIESVGLYWKLLTSLLKAIPERA 2 Nucleotide sequence of HCYJ-13 CTGCGTGCACTGCGCGACCGTTTCCAGCAGCGTGGTCAACAACTGCCGGTTCAAACTGATGCTTTCGATATGCACCTGCTGATTCGTCGTGTCCGCGCAGGCGATGGTTGGCCGGACCGTTACCACGTTCACGTATGGATCCGTCTGTTCGAACAGACCGCATTCCAGGCGGGTGTTGACCACCATGACGTGCGTCGTCTGGTGGAGCAGATCCTGATCCAGCTGGCGCACCAGGTCCATCACCGCCGCGTTTCTGTTCGTCTGCCTGCGCGTGTGGCAGCACTGCGTCGTCATCTGGGTGCTGGTCAGCTGGCCCAATGCGGCCAGCACCTGGACCACGTGATCTTCGCAGCGGTAGATCAACGTGCCGACCAGGCCGACGACTTCCGTTTCGTTTTTGCGGGTGGTGACCACGCGGATGTCCAACGTGGCTTTCATTACGCGTTCGACCGTGTTGCCCATGCTGACCATGCTGTTCGTCGTGCTGTTCAGCAGCGTCAGGAGGCTCTGCTGGCTGTTGGCGCGCAGTGCCTGCGTAGCGCCGGCGGTCGTCTGCAACAGCACGGTGACGTGCTGCTGTTCGGTGGTGAATTCGTGCTGCAGCATCGTCAGGAGCTGCTGGCCCAGCGCTTCCAGCGCGTTGGCTTCTTCCGCCGCCACCGTAATCACCGTGGCGGCTTTACGCGTAACGGTGTGGCACAAGGTGCGCCGGTGCAGATCCAGCACGCGCAGGTCCAGCGTCGCCGCATGGGCGAACAGGAAGCTCGTCAGCAATTCGTTGGCGTAGCACAGCCGGACGTTGACCTGGGCGCGGGTGTTGCGGCGTTCCAAGCTCTGGAGGGTCAGCTGCAGGGTCTGATTACCGGCCGTCATCGCTTCGCTCTGCAGCGCCAGGGTGGCGATGAGGTCGATACTGCTGGCGCAACCCACGTAGACTTTGCGTTCTTTTTCGGCATCGGCATCGACCAAGACGTCTGCCTGCAGCCGGTTTGGCTGCAGCCAGAAGGTGCAGTTCACGCGGGCTTCTTCGGTCACGGTCAACAGCACTTTCAACGTGCTGTCCTGAACGCGGTAATCGGTCAGCACCGCCAAGGTGGCGGTCACGCTGACGCCGTTGTTGGTACCCAGCGTGGTGCAGCGCGTTTCCATCCGCTGGCCGGCGACATCCGTCTGGATCGCATCCTGGGCGAAGTTGTTCACGGCGTCGTTATTCTGCTGCGCCATCACGTGCAAGTTCGTCTGCAGCGTCACCGTCTGGCAGTCTTCCATGCCTTCGGCGGTCGTTTCGCTCAGCAGGACATTGCCGATCTGATTGCGCTGGGTGTTCAGGTGCTGCTGTTCGGCCCGGGCGAAGATGTGCTGCGCCAGCGCTTTTTCATGATCCGTCGCGTACGCGACCGTACGGATCTGGGTGAAAACATCCCGCAGCGTCTGTGGCGCAAACTGCGTCAGTTTCGTCAT DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0035] Example 1: Preparation of aminopeptidase, comprising the following steps:

[0036] S1. Search and screen dipeptidases in the database. Specifically, by searching for β-Ala-His dipeptidase in the database, we obtained dipeptidase genes from different bacterial species, downloaded their sequences, and constructed a phylogenetic tree. After reviewing the information for the aforementioned enzymes, we identified the target genes as SmPepD, CpPepD, AnPepD, BmPepD, BtPepD, and HhPepD. The gene sequences were codon-optimized and synthesized by a biotechnology company.

[0037] S2. The dipeptidase encoding gene of step S1 is introduced into the receptor through plasmid construction and transformation, and the suitable template enzyme is screened by detecting the enzymatic activity of the dipeptidase. The specific steps are as follows:

[0038] (1) Plasmid construction: The pET28a plasmid was cut by Hind III and EcoR I restriction enzymes, and then the target gene fragment was connected to the cut plasmid by homologous recombination to form a recombinant plasmid. The recombinant plasmid containing the target gene was introduced into Escherichia coli BL21 (DE3) for heterologous expression.

[0039] (2) Transformation: Take out the competent E. coli cells from the -80℃ ultra-low temperature freezer and place them on ice for 3 minutes to cool and thaw. Add the recombinant plasmid to the competent cells and gently pipette to mix. Cool on ice for 10 minutes. Mix the plasmid and competent cells and transfer them to a pre-cooled electroporation cup. After standing on ice for 5 minutes, wipe the outside of the electroporation cup clean and place it in an electroporator. Cycle at 2000 V for 5 ms. After taking it out, quickly add 1 ml of LB medium as a recovery solution and recover at an appropriate temperature for 3 hours to 4 hours. Centrifuge at 4000 r / min for 5 minutes, discard the supernatant, and repeatedly pipette the remaining 100 μL of bacterial solution evenly. Spread it evenly on an LB plate containing 50 mg / L kanamycin and culture at an appropriate temperature overnight.

[0040] (3) Verification: The whole-cell reaction method was used to detect the synthetic activity of dipeptidase. A 200 μL reaction system included 50 mM Tris-HCl buffer, 1 M β-Ala, 50 mM L-His, and 8 mg of wet cells. The reaction was then stirred at 37°C and 200 rpm for 20 min. 40 μL of the reaction solution was taken and quenched by adding 760 μL of perchloric acid solution (pH 1.0). After mixing, the solution was filtered through a 0.22 μm aqueous membrane. The concentration of L-Car was detected by HPLC combined with an external standard method. The enzyme activities of the dipeptidase were compared. The enzyme with the highest synthetic activity, SmpPepD, was selected as the template enzyme for subsequent experiments.

[0041] Next, the molecular modification of dipeptidase was carried out as follows:

[0042] Alphafold 2.3.1 was used to predict the protein's structural model. PyMOL was used to analyze changes in the molecular tertiary structure. SmPepD and its mutants served as receptors, and small molecule products served as ligands. Schrödinger Maestro 12.8 was used for ligand docking, identification of the active center, and molecular dynamics simulations. Results were visualized in PyMOL. ESPript 3.0 was used for multiple sequence alignment (MSA). Evolutionary information from the position-specific scoring matrix (PSSM) was analyzed to illustrate the conservation of these protein residues and identify mutation sites. Ten mutation sites, including P81, T111, N116, M149, G151, D169, E171, L432, P451, and I453, were selected for mutation.

[0043] After identifying the mutation sites, 30 pre-mutants were selected based on PSSM scores. Recombinant mutants were constructed in E. coli BL21, induced for expression, and purified for subsequent enzyme activity assays. To further improve enzyme activity, the mutants were combined in pairs to construct double mutants in E. coli BL21. These mutants were induced for expression and purified for subsequent enzyme activity assays. Among the two point mutants, SmPepDT111S / G151D exhibited significantly higher enzyme activity than the wild type. This mutant, designated HCYJ-13, exhibited the highest enzyme activity.

[0044] The nucleotide sequence is as follows:

[0045]

[0046] The amino acid sequence is as follows:

[0047] MSELSQLSPQPLWDIFAKICSIPHPSYHEEALAQHILTWAKEKNLHAERDQVGNILLRKPATKGMENRKPVALQAHLDMVPQKNNDTVHDFAKDPIQPYIAGEWVKARGTSLGADNGIGMA SALAVLADDSVEHGPLEVLLTMTEEAGMDDAFGLQPNWLQADILINTDSEEEGEIYMGCAGGIDFITTLPLQREAVPAGYQTLKLTLKGLKGGHSGAEIHVGLGNANKLLARFLFAHAAALN LRVLDLNGGTLRNAIPREASAVVAVPADKADALKALSQEFLAVLQNELSAKEKNITVLLEPTTSASLALSADSQQRFLALLNGTPNGVIRMSDAVKGVVETSLNVGVVTTSENEAEIICLI RSLIDSGKDYVVEMLTALGQLAGAKVAPKGGYPGWQPDADSPVMHLVRELYQDLFNKTPNIMVIHAGLECGLFKKPYPNMDMVSIGPTITGPHSPDEQVHIESVGLYWKLLTSLLKAIPERA

[0048] Example 2, preparation of L-carnosine

[0049] A preparation method of L-carnosine, its process is as follows Figure 1 As shown, it includes the following steps:

[0050] S1, inoculating and fermenting the above-mentioned aminopeptidase to obtain β-aminopeptidase as an auxiliary modification enzyme, using β-alanine and L-histidine as substrates to react and obtain L-carnosine;

[0051] S2, the L-carnosine in step S1 is decolorized and then filtered through an ultrafiltration membrane. The resulting ultrafiltration membrane clear solution is further purified by resin to obtain analytical solution 1 and analytical solution 2. The analytical solution 2 is then concentrated and crystallized after removing impurities through a nanofiltration membrane, and then dried after recrystallization and purification to obtain high-purity L-carnosine.

[0052] The detailed experimental steps are as follows:

[0053] 1. Fermentation preparation of β-aminopeptidase

[0054] Streak the aminopeptidase strain on YPD and incubate overnight at 30°C in an inverted incubator. Pick a single colony (1 mm in diameter) from the plate and transfer it to 50 ml of YPD liquid medium (10 g yeast powder, 10 g peptone, 10 g glucose, and dilute to 1 L with water). Incubate overnight (24 hours) at 30°C with shaking at 200 rpm until the OD600 reaches 4-5. Inoculate a 10% inoculum of the strain into a shake flask containing 300 ml of YPD liquid medium (1 L Erlenmeyer flask). Incubate at 30°C with shaking at 200 rpm. After approximately 24 hours, the OD600 reaches approximately 12. Once the fermentation medium is prepared, transfer it to a 30 L fermentor and sterilize it at 121°C for 30 minutes. Cool the mixture to 30°C and adjust the pH to 5.0 with aqueous ammonia. Inoculate the fermentor with the culture at a 5% inoculum. Adjust the rotational speed and aeration based on the dissolved oxygen level to maintain it above 30%. After about 24 hours of culture, wait for a sudden increase in dissolved oxygen (DO), reaching a wet weight of approximately 140 g / L. Start feeding the culture with a 50% (w / v) glycerol aqueous solution at a rate of approximately 15 ml / L of fermentation broth / hour. This rate is controlled to maintain dissolved oxygen above 30%. Once the bacterial OD value (OD) exceeds 30, adjust the glycerol feed rate to 20-25 ml / L of fermentation broth / hour. Simultaneously, add IPTG for induction for 2-4 hours at a rate of 30 mg / L. After 24 hours of induction, when the bacterial OD value reaches above 200-250, stop fermentation, centrifuge the cells, homogenize with a high-pressure homogenizer to produce enzyme, and collect the enzyme solution.

[0055] 2. L-Carnosine Conversion Steps

[0056] β-Aminopeptidase catalyzes the hydrolysis or aminolysis of amide bonds or peptide bonds containing β-amino acid residues. The mechanism is that the substrate and the enzyme form an acylated intermediate, which then undergoes a nucleophilic interaction with another amino acid to complete the hydrolysis or aminolysis reaction. Due to the enzyme's exceptional catalytic activity for β-amino acid peptide substrates, β-alanine methyl ester hydrochloride (manufactured by Hubei Hongxin Ruiyu Fine Chemical Co., Ltd.) and L-histidine (manufactured by Hebei Huayang Biotechnology Co., Ltd.) are used as substrates. The β-aminopeptidase-catalyzed reaction is conducted at a temperature of 25-40°C and a reaction time of 4-6 hours to produce an L-carnosine conversion solution. The conversion solution is primarily L-carnosine, with small amounts of unreacted β-alanine and L-histidine substrates present. Therefore, extraction and purification of the conversion solution is required.

[0057] The conversion reaction of L-carnosine is as follows Figure 2 shown.

[0058] 3. Decolorization step

[0059] 1-2% hydrochloric acid was added to the L-carnosine conversion solution to adjust the pH of the conversion solution to 3-4, and 1%-2% ZX-775 activated carbon (manufacturer: Jiangsu Zhuxi Activated Carbon Co., Ltd.) was added to the conversion solution. The solution was stirred and decolorized for 0.5-1.0 h at a decolorization temperature of 20-40°C. The activated carbon was removed by filtration through a microporous filter, and the decolorized clear solution was transferred to an ultrafiltration process for further treatment.

[0060] 4. Ultrafiltration membrane filtration steps

[0061] The decolorized liquid is filtered through a 3000Da ultrafiltration membrane at a filtration temperature of 20-40°C and a filtration pressure of 0.4-0.6 MPa. L-carnosine is present in the ultrafiltration membrane clear liquid, which then enters the resin process for further treatment.

[0062] 5. Resin step

[0063] The ultrafiltration membrane supernatant was adsorbed on LKC60 resin (manufactured by Amicogen (China) Biopharmaceutical Co., Ltd.) at a loading rate of 1.0 BV / h. Both carnosine and histidine were adsorbed on the resin. Desorption: 0.02-0.05% ammonia (manufactured by Shandong Hengchang Shengcheng Chemical Co., Ltd.) was used to completely desorb the L-histidine, yielding Desorption Solution 1. Subsequently, 0.5-1% ammonia was used to desorb the L-carnosine, resulting in Desorption Solution 2, which was then subjected to the nanofiltration step.

[0064] 6. Nanofiltration step

[0065] The analytical solution 2 passes through the nanofiltration membrane (80D membrane core), the filtration temperature is 10-40℃, and the filtration pressure is 1.8mpa. L-carnosine will be retained by the nanofiltration membrane and exist in the nanofiltration concentrate, and the nanofiltration concentrate is concentrated.

[0066] 7. Concentration and crystallization step

[0067] The nanofiltration concentrate in step 5 was concentrated under reduced pressure at 60-65°C to a content of 30%-35%. Anhydrous ethanol was added dropwise to the concentrate at 40-45°C. When the solution became turbid, the addition of anhydrous ethanol was stopped. The solution was stirred and crystallized at 40-45°C for 30 minutes. Ethanol was continued to be added dropwise until a total volume of 4-5 times the volume of the concentrate was added. The solution was stirred and crystallized for 2 hours, then filtered and the filter cake was rinsed twice with anhydrous ethanol. The solid was dried with air at 50-55°C to obtain crude L-carnosine.

[0068] 8. Crystallization purification step

[0069] Take the dried sample and add it to 2 times the mass of deionized water, stir to dissolve, raise the temperature to 40-45°C, add anhydrous ethanol (manufacturer: Tangshan Zhongrong Technology Co., Ltd.) dropwise into the solution until the solution becomes turbid, stop adding, keep warm and stir for 30 minutes, continue to add anhydrous ethanol for a total of 4-5 times the mass of the solution, keep warm and stir for 2 hours, filter, rinse twice with ethanol, and collect wet crystals.

[0070] 9. Dry packaging steps

[0071] The wet product was placed in a vacuum drying oven, heated at 50-60°C, with a vacuum degree of ≤-0.095 MPa, and dried for 12 hours. After being crushed and passed through an 80-mesh sieve, the product was weighed and sealed in a PE bag and an aluminum foil bag to obtain the finished L-carnosine. The yield of L-carnosine was 80-85%, and the purity of L-carnosine was >99%. The liquid phase detection spectrum of L-carnosine is shown below. Figure 3 shown.

[0072] 10. Treatment of L-histidine analysis solution

[0073] Resol 1 (primarily containing L-histidine) was concentrated under reduced pressure at 60-65°C to a concentration of 30%-35%. Four to five volumes of anhydrous ethanol (manufactured by Tangshan Zhongrong Technology Co., Ltd.) were added to the L-histidine concentrate at 40-45°C. Stir and crystallize for 2 hours, then filter. Rinse the filter cake twice with ethanol. The solid was air-dried at 50-55°C. After weighing, the solid was sealed in a PE bag and then an aluminum foil bag to obtain L-histidine.

[0074] Throughout this specification, terms such as "one embodiment," "some embodiments," and "specific embodiments" mean that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Claims

1. An aminopeptidase that catalyzes the synthesis of L-carnosine, characterized in that The amino acid sequence of the aminopeptidase is shown in SEQ ID NO:

1.

2. The aminopeptidase according to claim 1, characterized in that The nucleotide sequence of the aminopeptidase is shown in SEQ ID NO:

2.

3. A method for preparing L-carnosine, characterized in that, The steps include: S1, inoculating the aminopeptidase according to any one of claims 1 to 2 and fermenting to obtain β-aminopeptidase as an auxiliary modification enzyme, using β-alanine and L-histidine as substrates to react and obtain L-carnosine; S2, the L-carnosine in step S1 is decolorized and then filtered through an ultrafiltration membrane. The resulting ultrafiltration membrane clear solution is further purified by resin to obtain analytical solution 1 and analytical solution 2. The analytical solution 2 is then concentrated and crystallized after removing impurities through a nanofiltration membrane, and then dried after recrystallization and purification to obtain high-purity L-carnosine.

4. the preparation method of L-carnosine according to claim 3, is characterized in that, In step S2, the decolorization is performed using activated carbon.

5. The preparation method of L-carnosine according to claim 3, wherein In step S2, the analytical solution 1 is concentrated, crystallized, and then dried to obtain L-histidine.

6. The preparation method of L-carnosine according to any one of claims 3 to 5, characterized in that In step S2, the nanofiltration membrane is an 80D membrane core.

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