A method for producing alpha-amino acid ester acyltransferase and valacyclopeptide

Abstract

The application discloses an alpha-amino acid ester acyltransferase and a production method of valine-tryptophan dipeptide, and relates to the technical field of biology. The amino acid sequence of the alpha-amino acid ester acyltransferase is shown in SEQ ID NO. 4. By designing and modifying the alpha-amino acid ester acyltransferase, the application successfully obtains a mutant with significantly improved catalytic performance, and establishes an efficient and green enzymatic synthesis process of valine-tryptophan dipeptide. The process is carried out under mild conditions, L-valine methyl ester and L-tryptophan are used as substrates, and the mutant enzyme expressed by the engineering bacteria is used for catalysis, so that the yield of valine-tryptophan dipeptide reaches 22.42 g / L, which is 1.62 times of the original enzyme. The process effectively overcomes the shortcomings of traditional chemical synthesis and existing enzymatic process, has the outstanding advantages of simple steps, mild conditions, high product concentration and environmental friendliness, and provides a reliable and promising technical scheme for the industrial production of valine-tryptophan dipeptide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for producing α-amino acid ester acyltransferase and valine dipeptide. Background Technology

[0002] Valine dipeptide, commonly known as dipeptide-2 or eye peptide, is a dipeptide composed of two amino acids, valine and tryptophan, with the molecular formula C64. 16 H 12 N3O3, with a molecular weight of 303.356 g / mol, is a valine dipeptide that has garnered significant attention in the beauty and skincare industry. Its unique chemical structure and physiological activity offer numerous potential applications. First, it can condition the skin, participating in maintaining the integrity of skin cell structure. Through interaction with cell surface receptors, it regulates cell metabolism and function, thereby promoting healthy skin. Therefore, it is widely used in cosmetics, particularly in eye creams, anti-aging serums, face creams / lotions, and eye makeup. Its structure is similar to the lipid content of the epidermis, thus it is considered a natural moisturizing factor (NMF). Second, it can improve lymphatic circulation, positively impacting the lymphatic system by increasing lymphatic fluid flow, helping to drain excess water from the eyes and face, reducing puffiness and eye bags. Third, it can remove dark circles, primarily by inhibiting the activity of angiotensin-converting enzyme, improving the vasoconstriction of blood vessels around the eyes, and promoting blood circulation, thereby effectively lightening dark circles.

[0003] Common methods for synthesizing valine dipeptides include chemical synthesis and biosynthesis. Chemical synthesis is complex, with low yield, low atom utilization, and difficult wastewater treatment, which hinders the large-scale industrial application of valine dipeptide synthesis. Compared to chemical synthesis, the advantages of biosynthesis (i.e., whole-cell synthesis) include: (1) one-step reaction with fewer side reactions and simpler separation and purification; (2) green synthesis with high atom utilization and strong economic efficiency; and (3) simple wastewater treatment after product purification, which is conducive to large-scale application. Therefore, in-depth research on the biosynthesis of valine dipeptides is of great significance for improving synthesis efficiency, saving production costs, and promoting the industrial development of biosynthesis of valine dipeptides. Summary of the Invention

[0004] The purpose of this invention is to provide a method for producing α-amino acid ester acyltransferase and valine dipeptide, thereby solving the problems existing in the prior art. This invention obtains a mutant by site-directed modification of EAET-EY01, which improves the activity of the α-amino acid ester acyltransferase, thus increasing the conversion rate of enzymatic synthesis of valine dipeptide.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an α-amino acid ester acyltransferase, the amino acid sequence of which is shown in SEQ ID NO.4.

[0006] The present invention also provides the encoding gene of the above-mentioned α-amino acid ester acyltransferase.

[0007] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0008] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0009] The present invention also provides a recombinant microbial strain, comprising the above-described recombinant expression vector.

[0010] The present invention also provides the application of the above-mentioned encoding gene, recombinant expression vector or recombinant microbial strain in the preparation of the above-mentioned α-amino acid ester acyltransferase.

[0011] The present invention also provides the application of the above-mentioned α-amino acid ester acyltransferase in the catalytic synthesis of valine dipeptide.

[0012] The present invention also provides the application of the above-mentioned recombinant microbial strains in the catalytic synthesis of valine dipeptides.

[0013] This invention also provides a method for producing valcitriol dipeptide using an enzymatic method, comprising the following steps: The valine dipeptide was prepared by using L-valine methyl ester and L-tryptophan as substrates and the above-mentioned α-amino acid ester acyltransferase as a catalytic reaction.

[0014] This invention also provides a method for producing valine dipeptide using a whole-cell method, comprising the following steps: The valine dipeptide was prepared by using L-valine methyl ester and L-tryptophan as substrates and by catalyzing a reaction in whole cells of the aforementioned recombinant microbial strain.

[0015] The present invention discloses the following technical effects: By designing and modifying α-amino acid ester acyltransferases, this invention successfully obtained mutants with significantly enhanced catalytic performance and established a highly efficient and green enzymatic process for the synthesis of valine dipeptides. This process, conducted under mild conditions, uses L-valine methyl ester and L-tryptophan as substrates and utilizes a highly active mutant enzyme expressed by engineered bacteria for catalysis. The resulting valine dipeptide yield reached a maximum of 22.42 g / L, 1.62 times that of the original enzyme. It effectively overcomes the shortcomings of traditional chemical synthesis and existing enzymatic processes, possessing significant advantages such as simple steps, mild conditions, high product concentration, and environmental friendliness. This provides a reliable and promising technical solution for the industrial production of valine dipeptides. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the enzymatic reaction for synthesizing valcitripeptide. Figure 2 The liquid chromatogram of the reaction solution in Comparative Example 1; Figure 3 The liquid chromatography peak chromatogram of the reaction solution in Example 4; Figure 4 The liquid chromatography peak diagram of the reaction solution in Example 5; Figure 5 This is a standard curve of valine dipeptide; Figure 6 The molecular formula diagram of valine dipeptide; Figure 7 The mass spectrum of the valeric dipeptide in Example 4; Figure 8 Valine dipeptide in Example 4 1 H-NMR spectrum; Figure 9 Valine dipeptide in Example 4 13 C-NMR spectrum. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Escherichia coli ( Escherichia coli EAET-EY01 was deposited on March 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beifu West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30105. It has been disclosed in patent application No. 202410565899.5, entitled "A recombinant Escherichia coli producing α-amino acid ester acyltransferase and its construction method and application".

[0024] The pET-22b-(+)-EAET-EY01 plasmid has been disclosed in patent application No. 202410565899.5, entitled "A recombinant Escherichia coli producing α-amino acid ester acyltransferase and its construction method and application". The nucleotide sequence of the EAET-EY01 gene contained in the plasmid is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes (EAET-EY01 protein) is shown in SEQ ID NO.2.

[0025] Example 1 A site-directed mutagenesis was performed at amino acid position 238 of the EAET-EY01 protein, changing the codon encoding asparagine to a codon encoding leucine, thus obtaining the mutated EAET-EY02 gene. The nucleotide sequence of the EAET-EY02 gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4. The site-directed mutagenesis used the pET-22b-(+)-EAET-EY01 plasmid as a template, and the primers used were as follows: Forward primer: 5'-CCGATCAAAGACCTATACCGTTTCTACGCAAG-3' (SEQ ID NO. 5); Reverse primer: 5'-TAGGTCTTTGATCGGGTATTCAAAGCGTTTCG-3' (SEQ ID NO. 6).

[0026] The EAET-EY02 gene is ligated into the pET-22b-(+) plasmid, which is then introduced into DMT competent cells for plasmid extraction and sequencing verification, to obtain the recombinant plasmid pET-22b-(+)-EAET-EY02.

[0027] SEQ ID NO. 1: GTGAAGAAACTGACGCTGAAAGTCACGCTGCTGACCCTGCTGCTGGGTTCTACTGTTGGTTTCGCACAAGATGCTAAAGCAGATTCTGCTTACGTACGTGATAACTACGAGAAGATCGAGCAGGTCATCCCTATGCGTGATGGTACTAAACTGTTCACCGCTATCTACCAGCCAAAGGACAAGACCAAACAGTATCCGGTCCTGCTGAACCGTACCCCTTATACTGTCGCACCTTACGGTGTTAACGAATACAAGAAATCCCTGGGTAACTTCCCTACCGAGATGCGTGAGGGCTTTATCTTCGTGTACCAAGACGTTCGTGGCAAATGGATGTCTGAAGGTGAATTTGAAGACGTGCGCCCTATCAACCCAAGCAAATCCAAGAAGGCAATCGACGAAAGCACCGACACCTTCGACACTCTGGAATGGCTGGCTAAAAACCTGAAGAACTACACCAAGAAAGCTGGCATCTACGGTATCTCCTACCCAGGTTTCTATTCCACCATGAGCCTGGTTAACTCCCACCCAACTCTGAAAGCAGTGTCTCCACAGGCACCAGTTACTAACTGGTTCCTGGGCGATGATTTCCATCATAACGGTGTGCTGTTCCTGAACGATTCTTTCTCCTTCATGACCTTCTTCGGTGTGAAACGTCCGCAGCCGATTACTCCGGATAAAGGTCCGAAACGCTTTGAATAC CCGATCAAAGAC AAC TACCGTTTCTACGCAAG

[0028] SEQ ID NO.2: VKKLTLKVTLLTLLLGSTVGFAQDAKADSAYVRDNYEKIEQVIPMRDGTKLFTAIYQPKDKTKQYPVLLNRTPYTVAPYGVNEYKKSLGNFPTEMREGFIFVYQDVRGKWMSEGEFEDVRPINPSKSKKAIDESTDTFDTLEWLAKNLKNYTKKAGIYGISYPGFYSTMSLVNSHPTLKA VSPQAPVTNWFLGDDFHHNGVLFLNDSFSFMTFFGVKRPQPITPDKGPKRFEYPIKD N YRFYASGSVKELKDKYLQDNIKFYNDLFAHPDYDQFWQDRNVLPHLTNVQPAVMTVGGFFDAEDVYGAFETYKAIEKQNPKATNIMVAGPWYHGGWVRSNGSTFGDMQFASNTSEHYQQEIELPFFNYYLKDKGNFKPTEATIFITGSNEWKQFDAWPPKNVTTQKIYLQQNGKIAFNKTNTTTTFDEYVADPNSPVPYSGGVLETRSREYMVDDQRFASTRPDVMVYQSDILTEDITLAGPVINHLVVSTTGTDADYVVKLIDVYPENTPKFNNKLMAGYQNLIRAEIMRGKYRNSFSNPEAMVPNKETNVTYTMPDVGHTFKKGHRIMIQVQNSWFPLADRNPQQFMNVYEATSKDYLKQTQRIYHTSYIEIPVLK。

[0029] SEQ ID NO.3: GTGAAGAAACTGACGCTGAAAGTCACGCTGCTGACCCTGCTGCTGGGTTCTACTGTTGGTTTCGCACAAGATGCTAAAGCAGATTCTGCTTACGTACGTGATAACTACGAGAAGATCGAGCAGGTCATCCCTATGCGTGATGGTACTAAACTGTTCACCGCTATCTACCAGCCAAAGGACAAGACCAAACAGTATCCGGTCCTGCTGAACCGTACCCCTTATACTGTCGCACCTTACGGTGTTAACGAATACAAGAAATCCCTGGGTAACTTCCCTACCGAGATGCGTGAGGGCTTTATCTTCGTGTACCAAGACGTTCGTGGCAAATGGATGTCTGAAGGTGAATTTGAAGACGTGCGCCCTATCAACCCAAGCAAATCCAAGAAGGCAATCGACGAAAGCACCGACACCTTCGACACTCTGGAATGGCTGGCTAAAAACCTGAAGAACTACACCAAGAAAGCTGGCATCTACGGTATCTCCTACCCAGGTTTCTATTCCACCATGAGCCTGGTTAACTCCCACCCAACTCTGAAAGCAGTGTCTCCACAGGCACCAGTTACTAACTGGTTCCTGGGCGATGATTTCCATCATAACGGTGTGCTGTTCCTGAACGATTCTTTCTCCTTCATGACCTTCTTCGGTGTGAAACGTCCGCAGCCGATTACTCCGGATAAAGGTCCGAAACGCTTTGAATAC CCGATCAAAGAC CTA TACCGTTTCTACGCAAG

[0030] SEQ ID NO.4: VKKLTLKVTLLTLLLGSTVGFAQDAKADSAYVRDNYEKIEQVIPMRDGTKLFTAIYQPKDKTKQYPVLLNRTPYTVAPYGVNEYKKSLGNFPTEMREGFIFVYQDVRGKWMSEGEFEDVRPINPSKSKKAIDESTDTFDTLEWLAKNLKNYTKKAGIYGISYPGFYSTMSLVNSHPTLKAVSPQAPVTNWFLGDDFHHNGVLFLNDSFSFMTFFGVKRPQPITPDKGPKRFEYPIKD L YRFYASGSVKELKDKYLQDNIKFYNDLFAHPDYDQFWQDRNVLPHLTNVQPAVMTVGGFFDAEDVYGAFETYKAIEKQNPKATNIMVAGPWYHGGWVRSNGSTFGDMQFASNTSEHYQQEIELPFFNYYLKDKGNFKPTEATIFITGSNEWKQFDAWPPKNVTTQKIYLQQNGKIAFNKTNTTTTFDEYVADPNSPVPYSGGVLETRSREYMVDDQRFASTRPDVMVYQSDILTEDITLAGPVINHLVVSTTGTDADYVVKLIDVYPENTPKFNNKLMAGYQNLIRAEIMRGKYRNSFSNPEAMVPNKETNVTYTMPDVGHTFKKGHRIMIQVQNSWFPLADRNPQQFMNVYEATSKDYLKQTQRIYHTSYIEIPVLK.

[0031] Example 2 The recombinant plasmid pET-22b-(+)-EAET-EY02 is transformed into Escherichia coli to construct a recombinant strain, and the specific method is as follows: BL21(DE3) competent cells were removed from the -80℃ freezer and thawed on ice. The recombinant plasmid pET-22b-(+)-EAET-EY02 to be transformed was added, and the cells were incubated on ice for 30 min. At the same time, the water bath was turned on and adjusted to 42℃. After the ice bath, the competent cells containing DNA were removed from the ice and heat-shocked at 42℃ for 45 s. Then, the cells were quickly incubated on ice for about 2 min, and 200 μL of LB (preheated at 37℃ for 1 min) was added. The cells were then thawed at 37℃ and 240 rpm for 1 h. The cells were then spread on ampicillin-resistant LB plates and cultured overnight at 37℃ to obtain recombinant Escherichia coli EAET-EY02 producing α-amino acid ester acyltransferase.

[0032] Example 3 LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0033] SE medium: glycerol 10 g / L, tryptone 10 g / L, yeast extract 10 g / L, CaCl2·2H2O 1 g / L.

[0034] Preparation of α-amino acid ester acyltransferase cell suspension: The recombinant Escherichia coli EAET-EY02 was inoculated into 50 mL LB medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 14–16 h until the absorbance OD value reached a certain level. 600 The absorbance was 2.5–3.0. 2 mL of seed culture was added to 200 mL of SE medium and incubated at 37°C and 220 rpm for 4 h until the absorbance OD value reached 2.5–3.0. 600 2. Add IPTG inducer at a concentration of 0.6 mmol / mL, ferment at 15℃ for 12 h, centrifuge at 8000 rpm at 4℃ for 15 min, remove the supernatant, add distilled water to shake and wash, and centrifuge again to obtain bacterial cells, add Tris-HCl buffer at pH=8.5 to prepare a whole cell suspension of 0.1 g / mL.

[0035] Enzyme activity assay: 10 mL of Tris-HCl buffer containing 200 mmol / L Val-OMe.HCl and 200 mmol / L Trp was prepared. The pH was adjusted to 8.5 with NaOH, and 1 mL of whole-cell suspension was added. The mixture was reacted at 25°C for 5 min. 1 mL of the reaction solution was then heated in a 65°C water bath for 10 min. The concentration of valine dipeptide was determined by high-performance liquid chromatography (HPLC). At 25°C, the amount of enzyme required to catalyze the production of 1 μmol of valine dipeptide per minute from 1 mL of substrate reaction solution was defined as one unit of enzyme activity (U). The enzyme activity of EAET-EY02 in the above whole-cell suspension was 223 U / mL.

[0036] Example 4 EAET-EY02-catalyzed synthesis of valine dipeptide: (1) Solution preparation: Prepare a mixed solution of 142 mM L-valine methyl ester (L-Val-OMe) and 100 mM L-tryptophan (L-Trp) using Tris-HCl buffer at pH=8.5. Adjust the pH of the substrate solution to 8.5 using 20% ​​NaOH solution.

[0037] (2) Enzymatic reaction: The whole cell suspension prepared in Example 3 was added to make the enzyme activity in the reaction system 30 U / mL. The mixture was stirred at 27°C for 60 min and the pH of the reaction solution was kept within 8.5±0.05 to catalyze the synthesis of valeropeptide.

[0038] (3) Reaction termination: The reaction was terminated by heating at 65°C for 10 min.

[0039] The structural formula of valine dipeptide is shown in [link to structural formula]. Figure 6 The mass spectrum of the catalytically synthesized valine dipeptide is shown in the image below. Figure 7 , 1 H-NMR spectrum see Figure 8 , 13 C-NMR spectrum see Figure 9 .

[0040] Comparative Example 1 EAET-EY01-catalyzed synthesis of valine dipeptide: (1) Solution preparation: Prepare a mixed solution of 100 mM L-Val-OMe and 80 mM L-Trp using Tris-HCl buffer solution with pH=8.5. Adjust the pH of the substrate solution to 8.5 using 20% ​​NaOH solution.

[0041] (2) Enzymatic reaction: EAET-EY01 whole-cell suspension (prepared using Escherichia coli EAET-EY01 according to the method in Example 3) was added to make the enzyme activity in the reaction system 30 U / mL. The mixture was stirred at 27℃ for 90 min, and the pH of the reaction solution was maintained within 8.5±0.05 to catalyze the synthesis of valeropeptide (see the catalytic synthesis process in Example 3). Figure 1 ).

[0042] (3) Reaction termination: The reaction was terminated by heating at 65°C for 10 min.

[0043] Example 5 To further improve the conversion rate, a fed-batch method was adopted to synthesize valine dipeptide using the EAET-EY02 enzyme: (1) Solution preparation: Prepare a mixed solution of 60 mM L-Val-OMe and 100 mM L-Trp using Tris-HCl buffer with pH=8.5. Adjust the pH of the substrate solution to 8.5 with 20% NaOH solution.

[0044] (2) Enzymatic reaction: The whole cell suspension prepared in Example 3 was added to make the enzyme activity in the reaction system 30 U / mL.

[0045] (3) Batch feeding: 40 mM L-Val-Ome was added in batches at 20 and 40 min of the reaction, the reaction temperature was kept at 27℃, and the pH of the reaction solution was kept within 8.5±0.05.

[0046] (4) Reaction termination: After 60 min of reaction, take a sample and heat it at 65℃ for 10 min to terminate the reaction.

[0047] Example 6 The conversion rates of valine dipeptide synthesized in Examples 4, 5, and Comparative Example 1 were calculated by detecting the concentration of valine dipeptide, as follows: Chromatographic conditions for detecting valine dipeptide: amide column (4.6 mm × 250 mm, 5 μm); mobile phase: 0.05 M KH2PO4 (pH 4.0): acetonitrile = 35:65; flow rate: 1 mL / min; detection wavelength: 280 nm.

[0048] Sample pretreatment: Take 350 μL of the reaction solution from Example 4, Example 5, or Comparative Example 1 and add it to 650 μL of acetonitrile. Dilute the mixture 4-fold with Tris-HCl buffer, centrifuge to remove the precipitate, and filter the supernatant through a 0.22 μm filter membrane. The liquid chromatography peak chromatogram of the reaction solution from Comparative Example 1 is shown below. Figure 2 As shown. The liquid chromatography peak chromatogram of the reaction solution in Example 4 is shown below. Figure 3 As shown. The liquid chromatography peak chromatogram of the reaction solution in Example 5 is shown below. Figure 4 As shown. A 1 g / L valachromapeptide standard solution was prepared using Tris-HCl buffer, and after serial dilution, a standard curve was constructed. The valachromapeptide standard curve is shown below. Figure 5 As shown.

[0049] The yield and conversion rate of valproic dipeptide in Examples 4, 5 and Comparative Example 1 are shown in Table 1.

[0050] Table 1. Valerox dipeptide yield and conversion rate In summary, by designing and modifying α-amino acid ester acyltransferases, this invention successfully obtained mutants with significantly enhanced catalytic performance and established a highly efficient and green enzymatic process for the synthesis of valine dipeptides. This process, conducted under mild conditions, uses L-valine methyl ester and L-tryptophan as substrates and utilizes a highly active mutant enzyme expressed by engineered bacteria for catalysis, achieving a maximum valine dipeptide yield of 22.42 g / L, 1.62 times that of the original enzyme. It effectively overcomes the shortcomings of traditional chemical synthesis and existing enzymatic processes, possessing significant advantages such as simple steps, mild conditions, high product concentration, and environmental friendliness. This provides a reliable and promising technical solution for the industrial production of valine dipeptides.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An α-amino acid ester acyltransferase, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

4.

2. A gene encoding an α-amino acid ester acyltransferase as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 2 or 3.

5. A recombinant microbial strain, characterized in that, Includes the recombinant expression vector as described in claim 4.

6. The use of the encoding gene as described in claim 2 or 3, the recombinant expression vector as described in claim 4, or the recombinant microbial strain as described in claim 5 in the preparation of the α-amino acid ester acyltransferase as described in claim 1.

7. The application of the α-amino acid ester acyltransferase as described in claim 1 in the catalytic synthesis of valine dipeptide.

8. The use of the recombinant microbial strain as described in claim 5 in the catalytic synthesis of valine dipeptide.

9. A method for producing valachromatin dipeptide using an enzymatic process, characterized in that, Includes the following steps: The valine dipeptide was prepared by using L-valine methyl ester and L-tryptophan as substrates and catalyzing the reaction with the α-amino acid ester acyltransferase described in claim 1.

10. A method for producing valcitriol dipeptide using a whole-cell approach, characterized in that, Includes the following steps: The valine dipeptide was prepared by using L-valine methyl ester and L-tryptophan as substrates and by catalytic reaction with whole cells of the recombinant microbial strain described in claim 5.

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