Aminopeptidase for synthesizing L-carnosine and its application

Abstract

The invention discloses an aminopeptidase for synthesizing L-carnosine and an application thereof. The amino acid sequence of the aminopeptidase is shown in SEQ ID NO: 3 or 5. The aminopeptidase of the invention can catalyze L-histidine to synthesize L-carnosine with high selectivity in the presence of β-alanine methyl ester, has high enzyme activity, and has the value of industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to an aminopeptidase for synthesizing L-carnosine and application thereof. Background Art

[0002] L-carnosine, also known as β-alanyl-L-histidine, is a dipeptide obtained by condensing two amino acids, β-alanine and L-histidine. It is a natural antioxidant in vivo. As a natural dipeptide, carnosine exists in large quantities in the muscles and brains of mammals. In vivo, carnosine has pH buffering, antioxidant, anti-free radical and anti-aging effects. Clinically, it is used to reduce visual fatigue and treat cataracts; zinc-containing carnosine can be used to treat gastric ulcers. Compared with other antioxidants, L-carnosine has the advantages of strong antioxidant capacity, no toxic side effects, and a variety of physiological activities. It has broad application prospects in the fields of medicine, health care, hygiene, and beauty.

[0003] There are many methods for producing L-carnosine. Since carnosine is present in high levels in animal muscle, carnosine was extracted directly from animal tissues in the early days. This method has low yield and poor purity and has long been eliminated. In the traditional chemical synthesis method, the active groups of L-histidine and β-alanine must be activated and protected respectively, and the protective groups must be eliminated after the subsequent condensation reaction. Although this method has a high yield, the entire synthesis process has many reaction steps and requires highly toxic preparations such as hydrazine hydrate. The product L-carnosine does not allow hydrazine residues, so there are high requirements for the extraction and purification of the product. A variety of enzymatic synthesis methods have been reported, including the reverse hydrolysis synthesis of β-alanine and L-histidine catalyzed by dipeptidyl hydrolase, the acyl transfer reaction synthesis between β-alaninamide / alanine ester and L-histidine catalyzed by aminopeptidase, and the condensation reaction synthesis of β-alanine and L-histidine catalyzed by carnosine synthetase with ATP energy. The reaction catalyzed by dipeptidyl hydrolase is limited by the reaction balance and requires the addition of high concentration of β-alanine for the reaction. The maximum conversion rate of L-histidine is only about 30%. The reaction catalyzed by carnosine synthetase depends on ATP, which has cost issues. Compared with the above two types of enzymatic reactions, the reaction catalyzed by aminopeptidase does not require the addition of high concentration substrates, does not rely on ATP, has fewer reaction steps, and is more suitable for industrial applications.

[0004] Nevertheless, the currently reported aminopeptidases have low activity, many by-products, and complex product separation and purification, which seriously limit the practical application of this method. Therefore, it is necessary to develop aminopeptidases with high activity and high selectivity. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the prior art lacks an aminopeptidase with high activity and high selectivity that can be used for enzymatic synthesis of L-carnosine, and provides an aminopeptidase for synthesizing L-carnosine and its application. The aminopeptidase of the present invention can catalyze L-histidine to synthesize L-carnosine with high selectivity in the presence of beta-alanine methyl ester, and has high enzyme activity, and has the value of industrial application.

[0006] In order to screen an aminopeptidase with high enzyme activity that catalyzes the synthesis of carnosine from L-histidine, the inventors screened a large number of microbial sources through literature retrieval and database retrieval analysis, and compared the various aminopeptidase resources accumulated in the laboratory, and selected an aminopeptidase from Stictis urceolatum, which was named S1, and its amino acid sequence is:

[0007] MTSSLPSTPRQRVREALPTVYLGRYATGSKNSLTDVPGVLVSTQSIHESPNYPNAAPDAINTGVTTILPRKNWFRKACFAGIFRLNGTGEMTGSHFIDETGLLHTPIIITGTFGVGSALNGVYEYAIREHSDDKGKVNWGILPVVAETSDGFLHDFTKFAVTPQHVVRGIDEASSDSVREGNTGGGTGMICHQFKGGTGSSSRM VPADGGKTYTIGALVQANYGAMRDFKISGAPTGRLICEEQEPKMQEDPNDPELFAQSSTVFNPPQEDDNKDGSIIVILGTDAPLHPAQLRRLAKRATVGLSR VGGWGSNLSGDIFLAFSTSNELDVHAATTANKKVDPWLPQEATIQMIDDQTINALLEASADVVEEAILNAVFMAETMEGNGNKVDALDLGKVKKLMEKY (SEQ ID NO: 1)

[0008] The disadvantages of wild-type aminopeptidase S1 are low enzyme activity and many side reactions. When catalyzing the conversion of L-histidine, in addition to synthesizing carnosine, it also catalyzes the continued reaction of carnosine to generate impurities, resulting in a decrease in the yield of carnosine.

[0009] In order to solve this problem, the inventors tried to transform the wild-type aminopeptidase S1 to improve the enzyme activity, so that the activity of L-histidine and β-alanine methyl ester was improved. Through homology modeling, a homology model of S1 was constructed, including a computer-simulated protein 3D model, and molecular docking with the substrate carnosine was performed. Combined with bioinformatics analysis, the hot amino acids in the active pocket and some amino acid sites that may interact were determined. Through rational analysis and semi-rational design, these amino acid sites were subjected to site-directed mutagenesis to construct a mutant library using the means of directed evolution. A R85T / Q194V mutant was screened out, named M2 herein, and its amino acid sequence is SEQ ID NO: 3, and the activity of catalyzing the synthesis of carnosine is significantly higher than that of the wild type.

[0010] Subsequently, the inventors also attempted to continue to transform the mutant M2 in order to reduce its enzyme activity of catalyzing the synthesis of impurities of carnosine and β-alanine methyl ester while maintaining its enzyme activity of catalyzing the synthesis of carnosine, thereby facilitating the industrial production of carnosine. For example, amino acid substitution was performed at position 150. Histidine (H or His) was used to replace the original serine (S or Ser), and a (R85T, S150H, Q194V, I319E) mutant relative to the wild enzyme S1 was obtained, which greatly reduced the activity of M2 catalyzing the synthesis of impurities of carnosine, and the carnosine content in the product when it catalyzed the conversion of L-histidine reached more than 80%, and the generation of impurities was reduced. The R85T / S150H / Q194V / I319E mutant was named M4, and its amino acid sequence was SEQ ID NO: 5, which has application value in the industrial production of carnosine.

[0011] On this basis, the inventors completed the present invention.

[0012] The present invention solves the above technical problems through the following technical solutions.

[0013] A first aspect of the present invention provides an aminopeptidase, the amino acid sequence of the aminopeptidase is shown in SEQ ID NO: 3 or 5.

[0014] The second aspect of the present invention provides a polynucleotide, characterized in that the polynucleotide encodes the aminopeptidase as described in the first aspect.

[0015] In some embodiments, the sequence of the polynucleotide is as shown in SEQ ID NO: 4 or 6.

[0016] As is known to all, the expression results of the same nucleotide sequence in different microbial hosts are often very different. In order to efficiently express aminopeptidases in Escherichia coli, which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon optimized.

[0017] Codon optimization is a technique that can be used to maximize protein expression in organisms by increasing the translation efficiency of the gene of interest. Different organisms usually show special preferences for some codons of the same amino acid being encoded due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the composition of its respective genome tRNA library is reflected by optimizing codons. Therefore, in fast-growing microorganisms, low-frequency codons of amino acids can be used for the same amino acids but high-frequency codon replacement. Therefore, the expression of the DNA sequence of optimized codons is improved in fast-growing microorganisms.

[0018] For example, to express aminopeptidase and its mutant M4 in E. coli, the gene encoding the codon-optimized wild-type aminopeptidase SEQ ID NO: 1 can be SEQ ID NO: 2; the gene encoding the aminopeptidase mutant M4 SEQ ID NO: 3 can be SEQ ID NO: 4.

[0019] The third aspect of the present invention provides a recombinant expression vector, which comprises the polynucleotide as described in the second aspect.

[0020] The fourth aspect of the present invention provides a transformant, which expresses the aminopeptidase as described in the first aspect, or contains the polynucleotide as described in the second aspect, or contains the recombinant expression vector as described in the third aspect; the transformant is not an animal variety or a plant variety.

[0021] In some embodiments, the transformant is a eukaryotic cell or a prokaryotic cell.

[0022] In some embodiments, the eukaryotic cell is selected from Pichia pastoris and Saccharomyces cerevisiae; and the prokaryotic cell is selected from Bacillus subtilis and Escherichia coli.

[0023] Herein, the host of the transformant can be any microorganism suitable for expressing aminopeptidase, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0024] In some embodiments, the E. coli is E. coli BL21 (DE3).

[0025] When the aminopeptidase is expressed in E. coli, the encoding gene can be cloned into an appropriate vector, such as pET-28a.

[0026] The aminopeptidase of the present invention has only 406 amino acids and a clear structure, so it is easy for a person skilled in the art to obtain a polynucleotide encoding it, an expression cassette and a plasmid comprising the polynucleotide, and a transformant comprising the plasmid through genetic engineering construction methods well known to those skilled in the art.

[0027] The fifth aspect of the present invention provides a use of the aminopeptidase as described in the first aspect, the polynucleotide as described in the second aspect, the recombinant expression vector as described in the third aspect, or the transformant as described in the fourth aspect in the preparation of L-carnosine.

[0028] A sixth aspect of the present invention provides a method for preparing an aminopeptidase, the method comprising:

[0029] The transformant according to the fourth aspect is cultured, and the aminopeptidase is obtained from the culture.

[0030] In some embodiments, in the transformant, the polynucleotide encoding the aminopeptidase is synthesized in vitro or obtained by mutation of the original polynucleotide.

[0031] The seventh aspect of the present invention provides a method for preparing L-carnosine, the method comprising catalyzing an acyl transfer reaction of L-histidine by the aminopeptidase as described in the first aspect in the presence of β-alanine methyl ester.

[0032] In some embodiments, each 1 mL of the catalytic reaction system contains 8-12 mg L-histidine, 10-15 mg β-alanine methyl ester and 80-120 μL of aminopeptidase.

[0033] In some embodiments, during the catalysis, the reaction pH is 8.0-9.0 and the temperature is 40-60°C.

[0034] When used as a biocatalyst for producing carnosine, the aminopeptidase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths containing enzymes, enzymes fixed on carriers, etc.; the bacterial cell forms include living bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0035] In some embodiments, the aminopeptidase is used in a form selected from the group consisting of pure enzyme, crude enzyme solution, fermentation broth containing aminopeptidase, enzyme powder and immobilized enzyme.

[0036] In some embodiments, the crude enzyme solution is prepared by the following method: ice-bath ultrasonic disruption of resting cells expressing the aminopeptidase in a buffer solution, and high-speed centrifugation followed by collection of the supernatant.

[0037] In some embodiments, the buffer is PBS buffer.

[0038] In some embodiments, the volume ratio of the crude enzyme solution is 10-12%.

[0039] In this article, the terms "wild type", "wild enzyme", and "wild-type enzyme" have the same meaning, and all refer to the aminopeptidase S1 having an amino acid sequence as shown in SEQ ID NO: 1. Sometimes, for the sake of convenience, the wild-type aminopeptidase and its mutants M2, M3, M4, etc. may be collectively referred to as "aminopeptidase" in this article.

[0040] The mutations at some sites in the amino acid sequence of the wild enzyme are not single mutations, for example, the mutation at position 85 can be R85T, R85L or R85I, in which case it can be expressed as R85TLI. The R85T mutation refers to a mutation in which the arginine (R or Arg) residue at position 85 of the amino acid sequence of SEQ ID NO: 1 is replaced by threonine (T or Thr), and so on.

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive and progressive effects of the present invention are:

[0044] The aminopeptidase of the invention can catalyze the synthesis of L-carnosine with high selectivity and has high enzyme activity, and has the value of industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the HPLC test result of M4 catalytic reaction.

[0046] Figure 2 This is the HPLC test result of S1 catalytic reaction.

[0047] Figure 3 This is the HPLC test result of M2 catalytic reaction.

[0048] Figure 4 This is the optimal pH test result for M4.

[0049] Figure 5 This is the optimal temperature detection result of M4.

[0050] Figure 6 The reaction route diagram. DETAILED DESCRIPTION

[0051] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0052] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0053] Materials and methods

[0054] In the examples, the whole gene synthesis, primer synthesis and sequencing were all commissioned to Sangon Biotechnology Co., Ltd.

[0055] The molecular biology experiments in the examples include competent cell preparation, transformation, medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and D. W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0056] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0057] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 15-20 g / L agar powder).

[0058] HPLC determination conditions of substrate L-histidine, carnosine and impurities:

[0059] Instrument model: Agilent 1290 Infinity II; Column model: ZORBAX SB-Aq (4.6*150 mm 5μm) Mobile phase: 50 mM NaClO 4 , ultrapure water; flow rate: 1mL / min; column temperature: 25℃; detection wavelength: 210nm; detection time: 3.6 min; injection volume 1μL; L-histidine retention time: 1.766min; carnosine retention time: 2.097min; impurity retention time: 2.917min.

[0060] It should be noted that, for the convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments. For example, S1 can represent a wild aminopeptidase expression strain, a plasmid pET-28a-S1 number, a wild enzyme SEQ ID NO: 1 number, and a wild enzyme encoding gene SEQ ID NO: 2 number.

[0061] Example 1: Synthesis and construction of wild-type aminopeptidase gene

[0062] Mining aminopeptidase genes in the NCBI database, commissioning Sangon Biotech to optimize its codons and perform full gene synthesis, the synthesized aminopeptidase gene was connected to the pET-28a vector, with restriction sites NdeI and HindIII, and the plasmid was transformed into the host E. coli BL21 (DE3) competent cells, evenly spread on LB solid medium containing 50μg / mL kanamycin, and inverted in a 37℃ incubator overnight. Pick a single clone and inoculate it into LB liquid medium containing 50μg / mL kanamycin, and culture it at 37℃ overnight to obtain an expression strain containing the aminopeptidase gene.

[0063] Example 2: Construction and screening of aminopeptidase S1 mutant library

[0064] Saturation mutagenesis, especially amino acid replacement, was performed on some sites of wild-type aminopeptidase S1, including R85T, S150H, Q194V, I319E and other computationally selected sites to construct a mutant library.

[0065] Template: pET-28a-S1, M1, M2, M3, etc.

[0066] The primers used to construct some mutants such as M2 (R85T / Q194V) and M4 (R85T / S150H / Q194V / I319E) are shown in Table 1:

[0067] Table 1 Primer sequences

[0068]

[0069] PCR reaction system (25 μL): 1-50 ng template, 0.3 μL (10 μM) of a pair of upstream and downstream primers, 2 μL of dNTP, 1 μL of Fast Pfu, 5 μL of 5X Buffer, and sterile water to make up to 25 μL.

[0070] The PCR amplification procedure was as follows: (1) pre-denaturation at 95°C for 2 min; (2) denaturation at 95°C for 30 sec, (3) annealing at 58°C for 30 sec, and (4) extension at 72°C for 4 min. Steps (2) to (4) were repeated for 30 cycles, followed by extension at 72°C for 10 min. The PCR product was stored at 4°C.

[0071] The amplified PCR product was digested with Dpn I for 3h at 37°C, and the PCR product was recovered with a purification kit, then transferred to E.coli BL21 (DE3) competent cells, added with culture medium and revived at 37°C for 40-50min, spread on LB solid culture medium containing kanamycin, inverted and cultured at 37°C overnight, picked a single clone and sent it to the company for sequencing, and the sequencing results were viewed with SnapGene biological analysis software to view the peak graph of the sequencing results to determine the correct mutation of the amino acid at the mutation site. The amino acid sequence of mutant M2 is SEQ ID NO: 3, and the nucleotide sequence of the expressed gene is SEQ ID NO: 4; the amino acid sequence of mutant M4 is SEQ ID NO: 5, and the nucleotide sequence of the expressed gene is SEQ ID NO: 6.

[0072] A mutant library of more than 600 mutants including M2 and M4 was constructed.

[0073] Example 3: Expression and enzyme activity determination of aminopeptidase S1 and its mutants

[0074] 3.1 The expression and purification of wild-type aminopeptidase S1 includes the following steps.

[0075] The expression strain of S1 obtained in Example 1 was inoculated into 2 mL of LB liquid medium containing kanamycin, and cultured at 37°C and 220 rpm for 5-8 hours, and then inoculated into 100 mL of LB medium containing kanamycin at a 1% (v / v) inoculation amount. When OD600 reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced in a shaker at 25°C and 220 rpm for 20 hours. The bacteria were collected by centrifugation at 10000 rpm, and washed twice with 0.1 M, pH 8.0 potassium phosphate buffer to obtain resting cells, and then the obtained cells were resuspended with 0.1 M, pH 8.0 potassium phosphate buffer (containing 10 mM imidazole), and ultrasonically disrupted in an ice bath (power 60 W, start 1 s, stop 3 s, run 15 min), and the supernatant, which was the crude enzyme solution of aminopeptidase S1, was collected by high-speed centrifugation at 12000 rpm for 5 min.

[0076] 3.2 Express and purify mutants M2, M3, M4, etc. separately using the same method.

[0077] 3.3 Enzyme activity assay

[0078] In a 1mL reaction system, add 60mM L-histidine, 78mM β-alanine methyl ester, 100μL crude enzyme solution, 900μL 50mM potassium phosphate buffer at pH 8.0, shake at 30℃, 220rpm for 10min. Take 500μL of the reaction solution, add 20μL 3MHCl and shake for 5min, centrifuge, filter the supernatant, and detect by HPLC. Calculate the enzyme activity according to the standard curve of L-carnosine.

[0079] Enzyme activity is defined as: under specific conditions (30°C, pH 8.0), the amount of enzyme required to convert 1 micromole of substrate or generate 1 micromole of product within 1 minute is one activity unit (U).

[0080] Example 4: Enzyme-catalyzed reaction with L-histidine and β-alanine methyl ester as substrates

[0081] 4.1 Taking mutant M4 as an example, the reaction of L-histidine conversion catalyzed by each mutant in the mutant library was examined.

[0082] Reaction system 1mL: 9.3g / L L-histidine, 10.9 g / L β-alanine methyl ester, 14.7 U crude enzyme solution. Reaction temperature 30℃. HPLC monitoring reaction, reaction route as shown Figure 6 As shown, the results are Figure 1 shown.

[0083] Depend on Figure 1 It can be seen that mutant M4 can convert L-histidine, and about 99% of the substrate L-histidine is converted into carnosine. It is particularly important that L-histidine almost generates the only product carnosine, and the yield of its byproducts is reduced, indicating that mutant M4 has higher enzyme activity and substrate selectivity.

[0084] 4.2 Comparison of the catalytic reactions of mutant M4, wild-type enzyme S1, and mutant M2

[0085] The catalytic reactions of wild enzyme S1 and other mutants such as M4 were compared according to the method in 4.1. The HPLC test results are as follows: Figure 2 , Figure 3 As shown. Figure 2 It can be seen that wild-type S1 can catalyze L-histidine to produce carnosine and impurities, and the ratio of these two products is close to 2:1; Figure 3 It can be seen that the mutant M2 has increased carnosine production and reduced impurity production, with a conversion rate higher than 94% and a ratio of the two products close to 28:1; the mutant M4 further reduces impurity production and increases carnosine production, with a conversion rate higher than 99% and impurities accounting for only 0.2%, indicating that the mutant M4 can effectively increase carnosine production and is more suitable for industrial production.

[0086] Example 5: Optimization of catalytic reaction conditions of mutant M4

[0087] The inventors also optimized the catalytic reaction conditions of mutant M4, including reaction pH, reaction temperature, etc.

[0088] 5.1 Effect of different pH on the reaction

[0089] The reaction system in Example 5.1 was used to react with citric acid-HCl buffer (pH 4-6), potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 6-8), Tris-HCl buffer (pH 8-9), and glycine-sodium hydroxide buffer (pH 9-10). After 1 hour of reaction, samples were taken and tested by HPLC. The results were as follows: Figure 4 As shown, it can be seen that when the reaction is carried out at pH 8.5, the conversion rate is the highest, that is, the optimal reaction pH is 8.5.

[0090] 5.2 Effect of different temperatures on the reaction

[0091] Under the reaction condition of pH 8, the same reaction system was used to react at 10℃, 15℃, 20℃, 25℃, 30℃, 40℃, 50℃ and 60℃. Comparing the 8 reaction temperatures, it was found that there was no obvious difference in the reaction between 40℃ and 60℃, but the activity was higher than that at other temperatures, indicating that the optimal reaction temperature is within 40~60℃, and the activity becomes worse below 30℃ (see the results). Figure 5 ).

[0092] In summary, compared with wild aminopeptidase S1, the mutant M4 (SEQ ID NO: 6) constructed and screened by the present invention can specifically catalyze the reaction of L-histidine to produce carnosine, its substrate specificity and enzyme activity are significantly enhanced, the amount of impurities generated is significantly reduced, and the feasibility of industrial application is enhanced.

Claims

1. An aminopeptidase, characterized in that The amino acid sequence of the aminopeptidase is shown in SEQ ID NO: 3 or 5.

2. A polynucleotide, characterized in that The polynucleotide encodes the aminopeptidase according to claim 1.

3. The polynucleotide according to claim 2, wherein The sequence of the polynucleotide is shown in SEQ ID NO: 4 or 6.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 2 or 3.

5. A transformant, characterized in that: The transformant expresses the aminopeptidase according to claim 1, or comprises the polynucleotide according to claim 2 or 3, or comprises the recombinant expression vector according to claim 4; the transformant is not an animal variety or a plant variety.

6. The transformant according to claim 5, characterized in that The transformant is a eukaryotic cell or a prokaryotic cell.

7. The transformant according to claim 6, characterized in that The eukaryotic cell is selected from Pichia pastoris and Saccharomyces cerevisiae; the prokaryotic cell is selected from Bacillus subtilis and Escherichia coli.

8. The transformant according to claim 7, characterized in that The Escherichia coli is E. coli BL21(DE3).

9. Use of the aminopeptidase according to claim 1, the polynucleotide according to claim 2 or 3, the recombinant expression vector according to claim 4, or the transformant according to any one of claims 5 to 8 in the preparation of L-carnosine.

10. A method for preparing aminopeptidase, characterized in that: The method comprises: Cultivate the transformant according to any one of claims 5 to 8, and obtain the aminopeptidase from the culture.

11. The method according to claim 10, characterized in that In the transformant, the polynucleotide encoding the aminopeptidase is synthesized in vitro.

12. A method for preparing L-carnosine, characterized in that: The method comprises the step of catalyzing an acyl transfer reaction of L-histidine by the aminopeptidase as claimed in claim 1 in the presence of β-alanine methyl ester; Wherein, each 1 mL of the catalytic reaction system contains 8-12 mg L-histidine, 10-15 mg β-alanine methyl ester and 1-30 U aminopeptidase; In the catalysis, the reaction pH is 8.0-9.0 and the temperature is 40-60°C.

13. The method according to claim 12, characterized in that The aminopeptidase is used in a form selected from pure enzyme, crude enzyme solution, fermentation broth containing the aminopeptidase, enzyme powder and immobilized enzyme.

14. The method according to claim 13, characterized in that The crude enzyme solution is prepared by the following method: the resting cells expressing the aminopeptidase are subjected to ice bath ultrasonic disruption in a buffer solution, and the supernatant is obtained after high-speed centrifugation.

15. The method according to claim 14, characterized in that The buffer is PBS buffer.

16. The method according to claim 13, characterized in that The volume ratio of the crude enzyme solution is 10-12%.

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