L-carnosine, preparation method thereof and aminopeptidase

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to L-carnosine, a preparation method thereof and aminopeptidase. On the basis of self-made aminopeptidase, the preparation method of the L-carnosine is developed on the basis of a synthetic biology theory, and the high-purity L-carnosine is prepared. According to the preparation method of the L-carnosine, provided by the invention, the high-purity L-carnosine is obtained, meanwhile, the recovery and reutilization of the L-histidine can be realized, and the cost is effectively reduced. The preparation method is mild in reaction condition, short in synthetic route and environment-friendly, and the optical purity of the product is high.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology, and specifically relates to L-carnosine and 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 dipeptide obtained by the condensation of two amino acids, β-alanine and L-histidine, and is a crystalline solid. It was first discovered in beef in 1900 and is widely present in the brain, muscles and other tissues of mammals. It is a natural active dipeptide, also known as L-carnosine. L-carnosine has antioxidant effects and anti-aging functions. It has therapeutic effects on hypertension, heart disease, senile cataracts, ulcers, etc., and has anti-tumor and other biological activities. Adding carnosine to fresh meat during storage can improve the flavor of the meat and extend the shelf life. L-carnosine has been proven to remove reactive oxygen free radicals (ROS) and α-β unsaturated aldehydes formed by excessive oxidation of fatty acids in cell membranes during oxidative stress. Adding carnosine to cosmetics can delay skin aging. Compared with other antioxidants, L-carnosine has the advantages of strong antioxidant capacity, no toxic side effects, and multiple physiological activities. It has broad application prospects in the fields of medicine, health care, hygiene, and beauty. At present, the exploration of new physiological activities and mechanisms of L-carnosine is still a hot topic.

[0003] At present, the production methods of L-carnosine include two categories: chemical synthesis and biosynthesis. The preparation of L-carnosine by chemical synthesis generally requires complex protection-deprotection steps for the active groups of the substrates β-alanine and L-histidine, and the synthesis steps are cumbersome, the reaction conditions are harsh, the product yield is low, the pollution is large, and there are problems such as toxic reagent residues.

[0004] The biosynthesis method has the advantages of mild reaction conditions, short synthesis route, environmental friendliness, and high optical purity of the product. Therefore, how to obtain a high yield of L-carnosine has always been a difficult problem, and the most important thing is to develop an efficient enzyme that can catalyze the synthesis of carnosine. Summary of the invention

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

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

[0007] In a first aspect, the present invention provides an aminopeptidase for catalyzing the synthesis of L-carnosine, wherein 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 problem of enzymes in biosynthesis of L-carnosine.

[0009] Based on 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 coding gene in step S1 into the receptor by plasmid construction and transformation, and finally screening out a suitable template enzyme by detecting the enzyme 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] Based on the above technical solution, the present invention can also be improved as follows.

[0016] Further, in step S2, the receptor is strain Escherichia coli BL21.

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

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

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

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

[0021] Based on the above technical solution, the present invention can also be improved as follows.

[0022] Furthermore, in step S2, the decolorization is carried out by using activated carbon.

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

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

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

[0026] The beneficial effects of the present invention are that the above process can not only obtain L-carnosine, but also obtain high-purity L-carnosine, and can realize the recovery and reuse of L-histidine, which effectively reduces the cost. In addition, the process of the present invention has mild reaction conditions, a short synthesis route, is environmentally friendly, and has high product optical purity.

[0027] The fourth aspect of the present invention provides an L-carnosine prepared by 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 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: Description Sequence 1 Amino acid sequence of HCYJ-13 MSELSQLSPQPLWDIFAKICSIPHPSYHEEALAQHILTWAKEKNLHAERDQVGNILLRKPATKGMENRKPVALQAHLDMVPQKNNDTVHDFAKDPIQPYIAGEWVKARGTSLGADNGIGMASALAVLADDSVEHGPLEVLLTMTEEAGMDDAFGLQPNWLQADILINTDSEEEGEIYMGCAGGIDFITTLPLQREAVPAGYQTLKLTLKGLKGGHSGAEIHVGLGNANKLLARFLFAHAAALNLRVLDLNGGTLRNAIPREASAVVAVPADKADALKALSQEFLAVLQNELSAKEKNITVLLEPTTSASLALSADSQQRFLALLNGTPNGVIRMSDAVKGVVETSLNVGVVTTSENEAEIICLIRSLIDSGKDYVVEMLTALGQLAGAKVAPKGGYPGWQPDADSPVMHLVRELYQDLFNKTPNIMVIHAGLECGLFKKPYPNMDMVSIGPTITGPHSPDEQVHIESVGLYWKLLTSLLKAIPERA 2 Nucleotide sequence of HCYJ-13 CTGCGTGCACTGCGCGACCGTTTCCAGCAGCGTGGTCAACAACTGCCGGTTCAAACTGATGCTTTCGATATGCACCTGCTGATTCGTCGTGTCCGCGCAGGCGATGGTTGGCCGGACCGTTACCACGTTCACGTATGGATCCGTCTGTTCGAACAGACCGCATTCCAGGCGGGTGTTGACCACCATGACGTGCGTCGTCTGGTGGAGCAGATCCTGATCCAGCTGGCGCACCAGGTCCATCACCGCCGCGTTTCTGTTCGTCTGCCTGCGCGTGTGGCAGCACTGCGTCGTCATCTGGGTGCTGGTCAGCTGGCCCAATGCGGCCAGCACCTGGACCACGTGATCTTCGCAGCGGTAGATCAACGTGCCGACCAGGCCGACGACTTCCGTTTCGTTTTTGCGGGTGGTGACCACGCGGATGTCCAACGTGGCTTTCATTACGCGTTCGACCGTGTTGCCCATGCTGACCATGCTGTTCGTCGTGCTGTTCAGCAGCGTCAGGAGGCTCTGCTGGCTGTTGGCGCGCAGTGCCTGCGTAGCGCCGGCGGTCGTCTGCAACAGCACGGTGACGTGCTGCTGTTCGGTGGTGAATTCGTGCTGCAGCATCGTCAGGAGCTGCTGGCCCAGCGCTTCCAGCGCGTTGGCTTCTTCCGCCGCCACCGTAATCACCGTGGCGGCTTTACGCGTAACGGTGTGGCACAAGGTGCGCCGGTGCAGATCCAGCACGCGCAGGTCCAGCGTCGCCGCATGGGCGAACAGGAAGCTCGTCAGCAATTCGTTGGCGTAGCACAGCCGGACGTTGACCTGGGCGCGGGTGTTGCGGCGTTCCAAGCTCTGGAGGGTCAGCTGCAGGGTCTGATTACCGGCCGTCATCGCTTCGCTCTGCAGCGCCAGGGTGGCGATGAGGTCGATACTGCTGGCGCAACCCACGTAGACTTTGCGTTCTTTTTCGGCATCGGCATCGACCAAGACGTCTGCCTGCAGCCGGTTTGGCTGCAGCCAGAAGGTGCAGTTCACGCGGGCTTCTTCGGTCACGGTCAACAGCACTTTCAACGTGCTGTCCTGAACGCGGTAATCGGTCAGCACCGCCAAGGTGGCGGTCACGCTGACGCCGTTGTTGGTACCCAGCGTGGTGCAGCGCGTTTCCATCCGCTGGCCGGCGACATCCGTCTGGATCGCATCCTGGGCGAAGTTGTTCACGGCGTCGTTATTCTGCTGCGCCATCACGTGCAAGTTCGTCTGCAGCGTCACCGTCTGGCAGTCTTCCATGCCTTCGGCGGTCGTTTCGCTCAGCAGGACATTGCCGATCTGATTGCGCTGGGTGTTCAGGTGCTGCTGTTCGGCCCGGGCGAAGATGTGCTGCGCCAGCGCTTTTTCATGATCCGTCGCGTACGCGACCGTACGGATCTGGGTGAAAACATCCCGCAGCGTCTGTGGCGCAAACTGCGTCAGTTTCGTCAT DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with 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] Embodiment 1, the preparation of aminopeptidase comprises the following steps:

[0036] S1. Search and screen dipeptidases in the database. Specifically, by searching for β-Ala-His dipeptidase in the database, dipeptidase genes from different strains were obtained, sequences were downloaded and a phylogenetic tree was established. The information of the above enzymes was checked and the target genes were determined to be SmPepD, CpPepD, AnPepD, BmPepD, BtPepD and HhPepD. The gene sequences were synthesized by a biological company after codon optimization.

[0037] S2, introducing the dipeptidase coding gene in step S1 into the receptor by plasmid construction and transformation, and finally screening out a suitable template enzyme by detecting the enzyme 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 E. 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 min to cool and thaw. Add the recombinant plasmid to the competent cells, gently blow and mix, cool in ice for 10 min, mix the plasmid and competent cells and transfer them to a pre-cooled electroporation cup. After standing on ice for 5 min, wipe the outside of the electroporation cup clean and place it in an electroporator. Click at 2000 V for 5 ms. After taking it out, quickly add 1 ml LB culture medium as the recovery solution and recover at an appropriate temperature for 3 hr - 4 hr. Centrifuge at 4000 r / min for 5 min, discard the supernatant, repeatedly blow and pipette the remaining 100 μL of bacterial solution evenly, evenly spread it on an LB plate containing 50 mg / L kanamycin, and culture it at an appropriate temperature overnight.

[0040] (3) Verification: The whole-cell reaction method was used to detect the synthetic activity of dipeptidase. The 200 μL reaction system included 50 mM Tris-HCl buffer, 1 M β-Ala, 50 mM L-His and 8 mg wet bacteria. Then the reaction was stirred at 37 °C and 200 rpm for 20 min. Take 40 μL of the reaction solution, add 760 μL of perchloric acid solution (pH 1.0) to quench the reaction, mix well and filter with a 0.22 μm aqueous membrane, use HPLC combined with the external standard method to detect the concentration of L-Car and compare the enzyme activities of each dipeptidase, and select the enzyme with the highest synthetic activity, SmpPepD, as the template enzyme for subsequent experiments.

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

[0042] Alphafold2.3.1 was used to predict the structural model of the protein. The PyMOL program was used to analyze the changes in the tertiary structure of the molecule. SmPepD and its mutants were used as receptors, and small molecule products were used as ligands. SchrÖdinger Maestro 12.8 was used for docking with the ligand to determine the active center and molecular dynamics simulation. The results were observed in PyMOL. The program ESPript3.0 was used for multiple sequence alignment (MSA). The evolutionary information of the position-specific scoring matrix (PSSM) was analyzed to illustrate the conservation of these protein residues and determine the mutation sites. Ten mutation sites, including P81, T111, N116, M149, G151, D169, E171, L432, P451, and I453, were selected for mutation.

[0043] After the mutation site was determined, 30 pre-mutants were screened and determined based on the PSSM score. Recombinant strain mutants were constructed in E. coli BL21, induced expression and purification were performed for subsequent enzyme activity determination. In order to further improve the activity of the enzyme, the mutants were mutated in pairs, and double mutants were constructed in E. coli BL21, induced expression and purification were performed for subsequent enzyme activity determination. Among the two-point mutants, the enzyme activity of SmPepDT111S / G151D was significantly higher than that of the wild type, and it was named HCYJ-13, which had 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] Embodiment 2, the preparation of L-carnosine

[0049] A preparation method of L-carnosine, its process flow 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, decolorizing the L-carnosine in step S1, and then filtering through an ultrafiltration membrane, the obtained ultrafiltration membrane clear solution is further purified by a resin to obtain analytical solution 1 and analytical solution 2, and then the analytical solution 2 is 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] Take the original strain aminopeptidase and streak it on YPD, invert and culture it overnight at 30℃. Pick a single colony (1mm in diameter) on the plate and add it to 50ml YPD liquid medium (10g yeast powder, 10g peptone, 10g glucose, add water to 1L), shake and culture it at 30℃, 200rpm overnight (24h), and OD600 will increase to 4-5. Inoculate 10% of the inoculum into a shake flask containing 300mlYPD liquid medium (1L triangular flask), shake and culture it at 30℃, 200rpm, and OD600 will grow to about 12 after about 24 hours. After the fermentation medium is prepared, transfer it to a fermenter (30L), sterilize it at 121℃ for 30min; cool it to 30℃, and use ammonia water to adjust the pH value to 5.0. Inoculate the cultured bacterial liquid into the fermenter with an inoculum of 5%. Adjust the speed and ventilation according to the dissolved oxygen to control the dissolved oxygen above 30%. After about 24 hours of cultivation, when the dissolved oxygen suddenly rises and the wet weight is about 140g / L, start feeding 50% (w / v) glycerol aqueous solution at a rate of about 15ml / L fermentation liquid / hour. The feeding rate controls the dissolved oxygen to be above 30%. When the bacterial concentration OD is greater than 30, the glycerol supplement rate is adjusted to 20-25ml / L fermentation liquid / hour, and IPTG is added for induction for 2-4 hours at the same time. The amount of IPTG inducer added is 30mg / L. After 24 hours of induction, when the bacterial concentration OD reaches more than 200-250, stop fermentation, centrifuge the bacteria, homogenize the enzyme with a high-pressure homogenizer, and collect the enzyme solution.

[0055] 2. L-Carnosine Conversion Steps

[0056] β-aminopeptidase has the activity of catalyzing the hydrolysis or aminolysis reaction of amide bonds or peptide bonds containing β-amino acid residues. The mechanism is that the substrate and the enzyme form an acylated intermediate, and then react with another amino acid to complete the hydrolysis or aminolysis reaction. Because of the special catalytic activity of the enzyme 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 reaction substrates, and the reaction is catalyzed by β-aminopeptidase. The conversion temperature is 25-40°C, and the conversion reaction time is 4-6 hours to obtain L-carnosine conversion liquid. The main component of the conversion liquid is L-carnosine, and it also contains a small amount of unreacted β-alanine and L-histidine substrates. Therefore, the conversion liquid needs to be extracted and purified.

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

[0058] 3. Decolorization steps

[0059] Add 1-2% hydrochloric acid to the L-carnosine conversion solution to adjust the pH of the conversion solution to 3-4, add 1%-2% ZX-775 activated carbon (manufacturer: Jiangsu Zhuxi Activated Carbon Co., Ltd.) to the conversion solution, stir and decolorize for 0.5-1.0h, the decolorization temperature is 20-40℃, filter through a microporous filter to remove the activated carbon, and transfer the decolorized clear liquid to the ultrafiltration process for further treatment.

[0060] 4. Ultrafiltration membrane filtration steps

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

[0062] 5. Resin Steps

[0063] The ultrafiltration membrane clear solution was adsorbed by LKC60 resin (resin manufacturer: Amicogen (China) Biopharmaceutical Co., Ltd.), with a sample loading rate of 1.0BV / h, and both carnosine and histidine were adsorbed on the resin. Analysis: 0.02-0.05% ammonia water (manufacturer: Shandong Hengchang Shengcheng Chemical Co., Ltd.) was used for analysis, and L-histidine was completely analyzed to obtain analysis solution 1. Then 0.5-1% ammonia water was used for analysis, and L-carnosine was analyzed, and the obtained analysis solution 2 was sent to the nanofiltration step.

[0064] 6. Nanofiltration Steps

[0065] The analytical solution 2 passes through a nanofiltration membrane (80D membrane core), the filtration temperature is 10-40°C, 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 steps

[0067] The nanofiltration concentrate in step 5 is concentrated under reduced pressure at 60-65°C to a content (30%-35%). Anhydrous ethanol is added to the concentrate at 40-45°C. When the solution becomes turbid, the addition of anhydrous ethanol is stopped. The solution is stirred and crystallized at 40-45°C for 30 minutes. Ethanol is continued to be added, and a total of 4-5 times the volume of anhydrous ethanol is added. After stirring and crystallizing for 2 hours, the filter is filtered and the filter cake is rinsed twice with anhydrous ethanol. The solid is 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, stop adding when the solution becomes turbid, keep warm and stir for 30 minutes, continue to add anhydrous ethanol totaling 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, it was weighed and sealed with a PE bag and an aluminum foil bag to obtain the finished L-carnosine product. The yield of L-carnosine is 80-85%, and the purity of L-carnosine is >99%. The liquid phase detection spectrum of L-carnosine is as follows Figure 3 shown.

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

[0073] The analytical solution 1 (mainly containing L-histidine) was concentrated under reduced pressure at 60-65°C to a content of (30%-35%). At 40-45°C, 4-5 times the volume of anhydrous ethanol (manufactured by Tangshan Zhongrong Technology Co., Ltd.) was added to the L-histidine concentrate, stirred for 2 hours and then filtered, and the filter cake was rinsed twice with ethanol; the solid was dried at 50-55°C. After weighing, it was sealed with a PE bag and an aluminum foil bag to obtain L-histidine.

[0074] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "specific embodiments", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

Claims

1. An aminopeptidase for catalyzing 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 an aminopeptidase according to any one of claims 1 or 2, characterized in that: The steps include: S1. Search and screen dipeptidases in the database; S2, introducing the dipeptidase coding gene in step S1 into the receptor by plasmid construction and transformation, and finally screening out a suitable template enzyme by detecting the enzyme activity of the dipeptidase; 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.

4. The method for preparing aminopeptidase according to claim 3, characterized in that: In step S2, the recipient is the strain Escherichia coli BL21.

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

6. The preparation method of L-carnosine according to claim 5, characterized in that, In step S2, the decolorization is carried out by using activated carbon.

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

8. The preparation method of L-carnosine according to claim 5, characterized in that, In step S2, the resin is LKC60 resin.

9. The method for preparing L-carnosine according to any one of claims 5 to 9, characterized in that: In step S2, the nanofiltration membrane is an 80D membrane core.

10. An L-carnosine, characterized in that: The L-carnosine is prepared by the preparation method of any one of claims 5 to 9.

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