L-amino acid ligase and application thereof in catalytic synthesis of L-valine dipeptide and derivatives thereof

Abstract

The invention discloses an L-amino acid ligase and application thereof in catalytic synthesis of L-valine dipeptide and derivatives thereof, the amino acid sequence of the L-amino acid ligase is selected from any one sequence of SEQ ID No: 2-9, the L-amino acid ligase is prepared by firstly constructing a genetic engineering strain of a biological enzyme, and synthesizing a biological enzyme gene segment obtained by whole-gene synthesis into L-valine dipeptide and a derivative of the L-valine dipeptide. Using restriction endonuclease to linearize recombinant plasmids, and then performing activation and fermentation induction culture on the positive clones to prepare the recombinant plasmid. The invention also provides a method for catalytically synthesizing the L-valine dipeptide and the derivative thereof by using the L-amino acid ligase disclosed by the invention, the synthesis route is simple, the raw materials are cheap and easy to obtain, the use amount of the enzyme is less than 0.5%, the cost is low, and the conversion rate can reach more than 80%. The method has the advantages of simple post-treatment process, high product purity, high safety and small environmental pollution, and has extremely high economic value and industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an L-amino acid ligase and its application in catalytic synthesis of L-valine dipeptide and its derivatives. Background Art

[0002] In recent years, peptide-based nanomaterials have shown great application potential in materials science, tissue engineering, bioengineering, and drug delivery due to their good biocompatibility and diversity of structure and function. In the prior art, a series of peptide-based nanostructures can be constructed using dipeptides as self-assembly units. These chemically modified dipeptides can be assembled as modular units of peptide-based nanomaterials to achieve the effect of drug delivery. As a result, assembled peptide nanostructured materials show their unique advantages in application.

[0003] The current methods for synthesizing dipeptides are mainly chemical synthesis (solid phase method, liquid phase method) and enzyme synthesis. Chemical synthesis of dipeptides often requires the protection of certain functional groups of amino acids, and then the target group is attached after deprotection. The steps are complicated and require the use of a large amount of organic solvents. The final product may also contain racemates. For example, in the prior art, patent CN102199194A discloses a method for chemical synthesis of dipeptides and their derivatives. The whole process requires the chlorination of one of the amino acids in the raw material. After the intermediate product is generated, SOCl is added under ice-salt bath conditions. 2 Prepare amino acid hydrochloride, wait for the reaction to end, add strong base for deprotection, and obtain the final target product; Patent CN102199194A discloses a method for chemically synthesizing leucine dipeptide and its derivatives, and the whole process also requires adding tert-butyloxycarbonyl and benzyl protection to both ends of the product. Therefore, chemical synthesis is not an ideal method that is economical, efficient, and environmentally friendly.

[0004] L-amino acid ligase (Lal) is the main catalytic enzyme for the existing enzymatic synthesis of dipeptides. L-amino acid ligase (Lal) can connect two free amino acids to synthesize dipeptides under the participation of ATP. However, its substrate spectrum is narrow, and mainly natural amino acids are used to synthesize dipeptides. There are few reports on the existing L-amino acid ligase synthesizing non-natural dipeptides. For example, patent CN116622795A discloses a method for enzymatic synthesis of dipeptides composed of natural amino acids. The invention expands the substrate spectrum of L-amino acid ligase, but does not involve the synthesis of dipeptide derivatives of non-natural amino acids. This severely limits the widespread application of peptide-based nanomaterials with multiple functions. Therefore, the development of L-amino acid ligases for non-natural amino acids is of great significance to the modular construction of synthetic dipeptides and peptide-based nanomaterials.

[0005] Therefore, the art is in urgent need of an L-amino acid ligase and its application in the catalytic synthesis of L-valine dipeptide and its derivatives, which is suitable for the synthesis of dipeptides of non-natural amino acids and the construction of peptide-based nanomaterials. At the same time, the method for catalytic synthesis of dipeptides and their derivatives is easy to operate, low in cost and high in yield, and has excellent economic value and practicality. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an L-amino acid ligase, wherein the amino acid sequence of the L-amino acid ligase comprises at least any one of the following sequences:

[0007] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 2; or

[0008] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 3; or

[0009] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 4; or

[0010] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 5; or

[0011] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 6; or

[0012] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No:7; or

[0013] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 8; or

[0014] An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No:9.

[0015] Specifically, the L-amino acid ligase is derived from Bacillus subtilis, Pseudomonas, Ralstonia solanacearum, Yersinia, Streptomyces, Bacillus velezensis, Bacillus thuringiensis or Bacillus thuringiensis.

[0016] Specifically, the L-amino acid ligase is used to catalyze the synthesis of L-valine dipeptide and its derivatives. The L-amino acid ligase is prepared by a genetically engineered strain constructed by in vitro recombination, and the genetically engineered strain is Escherichia coli BL21, BL21 (DE3) and BL21Star (DE3).

[0017] In a second aspect, the present invention provides a nucleic acid encoding an L-amino acid ligase, wherein the nucleic acid comprises a nucleotide sequence having at least 75% identity with the sequence shown in SEQ ID No:1.

[0018] Specifically, the L-amino acid ligase encoded by the nucleic acid is derived from Bacillus subtilis.

[0019] In a third aspect, the present invention provides a method for preparing an L-amino acid ligase, wherein the method is used to prepare the aforementioned L-amino acid ligase, and the method comprises the following steps:

[0020] 1) Construction of genetically engineered strains: The nucleotide sequence of L-amino acid ligase is digested and recombined into an expression vector, which is transformed into a host cell and plated on an LB solid agar plate to obtain an engineered strain containing the L-amino acid ligase gene;

[0021] 2) selecting the positive clones of the genetically engineered strain obtained in step 1) and activating them in LB liquid medium, and then inoculating them in LB fermentation medium for culture, inducing expression, and obtaining an expression host containing the L-amino acid ligase;

[0022] 3) The expression host obtained in step 2) is ultrasonically disrupted and centrifuged to obtain a supernatant, and the obtained supernatant is ultrafiltered, concentrated, and then freeze-dried to obtain a freeze-dried enzyme powder of L-amino acid ligase.

[0023] Specifically, in an aqueous phase system, an L-valine analog reacts with L-valine under the action of L-amino acid ligase, ATP and metal ions to generate L-valine dipeptide and its derivatives, wherein the nucleotide sequence of the L-amino acid ligase is a nucleotide sequence having at least 75% identity with the sequence shown in SEQ ID No: 1, and the amino acid sequence of the L-amino acid ligase is an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID No: 2.

[0024] Specifically, the L-valine analog is selected from any one of L-valine, N-methyl-L-valine, N-ethyl-L-valine, N,N-dimethyl-L-valine or N-acetyl-L-valine.

[0025] Specifically, the pH of the aqueous phase system is 6.0-8.0, and the aqueous phase system is selected from any one of phosphate buffer, HEPES or Tris-HCl buffer.

[0026] Specifically, the mass concentrations of the L-valine analog and L-valine are both 0.1-100 mg / mL.

[0027] Specifically, the ATP is in the form of ATP or its salt ions, and the mass concentration of the ATP is 0.1 to 100 mg / mL.

[0028] Specifically, the metal ion is selected from any one of magnesium chloride, manganese chloride or cobalt chloride, and the mass concentration of the metal ion is 0.1 to 100 mg / mL.

[0029] Specifically, the L-amino acid ligase is L-amino acid ligase freeze-dried enzyme powder, and the amount of the L-amino acid ligase freeze-dried enzyme powder used is 0.1-10% of the amount of the L-valine analog.

[0030] Specifically, the reaction temperature is 25 to 40° C., and the reaction time is 12 to 24 hours.

[0031] Specifically, the method comprises the following steps:

[0032] S1, adding L-valine analogs and L-valine to an aqueous phase system, and then adding ATP and metal ions;

[0033] S2, adjusting the pH of the reaction system to 6.0-8.0 using NaOH, adding L-amino acid ligase and maintaining the pH of the reaction system at 6.0-8.0;

[0034] S3, raising the reaction temperature to 25-40°C and reacting for 12-24 hours;

[0035] S4, raising the reaction temperature to 90°C to inactivate the enzyme;

[0036] S5, adding a derivatization reagent to the reaction solution for derivatization;

[0037] S6. Use UHPLC ultra-high performance liquid chromatography to analyze and detect the reaction conversion rate.

[0038] Specifically, the derivatization reagent is Fmoc-Cl.

[0039] The beneficial effects of the present invention are as follows:

[0040] 1) The L-amino acid ligase disclosed in the present application can be used for the synthesis of non-natural amino acid dipeptides. It can be used as a catalyst to enzymatically synthesize L-valine dipeptide and its derivatives. After heating inactivation and filtration, nanofiltration technology is used to remove the generated salt. After concentration, methanol and water are used for crystallization to obtain high-purity L-valine dipeptide and its derivatives.

[0041] 2) The synthetic route disclosed in the present application is simple, the raw materials are cheap and easily available, the equipment requirements are simple, and the enzyme dosage is small (1%); the yield is high, the post-treatment process is simple, and the product purity is high; it is highly safe, has little pollution to the environment, is low in cost, and has extremely high economic value and industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the UHPLC result spectrum of the enzymatic synthesis of L-valine-L-valine in the present invention;

[0043] Figure 2 This is the UHPLC result spectrum of the enzymatic synthesis of N-methyl-L-valine-L-valine in the present invention;

[0044] Figure 3 This is the UHPLC result spectrum of the enzymatic synthesis of N-ethyl-L-valine-L-valine in the present invention;

[0045] Figure 4 This is the UHPLC result spectrum of the enzymatic synthesis of N,N-dimethyl-L-valine-L-valine in the present invention;

[0046] Figure 5 This is a spectrum of UHPLC results of enzymatic synthesis of N-acetyl-L-valine-L-valine in the present invention. DETAILED DESCRIPTION

[0047] The present application discloses L-amino acid ligases derived from Bacillus subtilis, Pseudomonas syringae, Ralstonia solanacearum, Yersinia intermedia ATCC 29909, Streptomyces sp.SceaMP-e96, Bacillus velezensis, Bacillus altitudinis and Bacillus thuringiensis, and discloses a preparation method thereof and a method for synthesizing L-valine dipeptide and its analogs catalyzed by the L-amino acid ligase. The structural formula of the reaction is as follows:

[0048]

[0049] In the formula, R 1 =H, R 2 =H; or

[0050] R 1 =H,R 2 =CH 3 ;or

[0051] R 1 =H,R 2 =CH 2CH 3 ;or

[0052] R 1 =CH 3 ,R 2 =CH 3 ;or

[0053] R 1 =H, R 2 =COCH 3 .

[0054] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Unless otherwise specified, the raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0055] The present invention is further described below in conjunction with a plurality of examples. It should be understood that these examples are merely illustrative and are not intended to limit the present invention. The methods, materials and reagents used in the examples are, unless otherwise stated, conventional methods in the art, and materials and reagents available from commercial sources.

[0056] Example 1 Construction and transformation of recombinant vector

[0057] Taking the construction of recombinant strain E. coli BL21 (DE3) pET-28a-Lal as an example

[0058] Activate E. coli DH5α / pET-28a(+) strain (E. coli DH5α purchased from Novizan Biotechnology Co., Ltd.), add 3-5 μL pET-28a-Lal (50 ng / μL) to 50-100 μL E. coli BL21(DE3) competent cells (purchased from Novizan Biotechnology Co., Ltd.), place on ice for 20 min, heat shock at 42°C for 90 sec, quickly return to ice for 5 min, add 800 μL of resistance-free LB culture solution, incubate at 37°C, 200 rpm, culture for 1 h, then spread on LB agar plate medium containing kanamycin resistance, and culture overnight at 37°C for 12 h to obtain recombinant E. coli BL21(DE3) containing pET-28a-Lal.

[0059] Among them, L-amino acid ligase (Lal) is derived from Bacillus subtilis, the nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0060] The formula of the non-resistant LB culture medium is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L;

[0061] The formula of LB agar plate medium containing kanamycin resistance is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, agar powder 20g / L, kanamycin 25μg / mL.

[0062] Example 2 Construction and expression of expression vector

[0063] Taking the construction and expression of E. coli BL21 (DE3) recombinant bacteria containing pET28a-Lal as an example

[0064] The transformed recombinant E. coli BL21 (DE3) containing pET28a-Lal was directly spread on a solid LB plate containing 25 μg / mL kanamycin resistance and cultured at 37°C for 12-14 hours to obtain a monoclonal colony. A single colony of E. coli BL21 (DE3) containing the Lal recombinant vector was picked from the kanamycin plate and inoculated into 1 mL of liquid LB medium containing 25 μg / mL kanamycin resistance and cultured at 37°C for 12 hours. Then, the cells were inoculated at a 2% (v / v) inoculum into 1 L of fresh liquid LB medium containing 25 μg / mL kanamycin resistance, cultured at 37°C until OD600 was approximately 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L, induced at 200 rpm, and 25°C for 20 h, then centrifuged (4°C, 4000 rpm, 30 min) to remove the supernatant, and the obtained bacterial sludge was washed with 30 ml of 0.9% NaCl solution and resuspended for later use.

[0065] Example 3 Preparation of L-amino acid ligase (Lal) lyophilized enzyme powder

[0066] The collected bacterial liquid was washed twice with 50mmol / L Tris-HCl, and then resuspended in 50mmol / L Tris-HCl (pH8.0) buffer, and the cells were ultrasonically disrupted in an ice bath (amplifier 6, power 500W, on 2s, off 5s, 30 minutes). The ultrasonically disrupted sample was centrifuged at 12000rpm and 4℃ for 30min, and the supernatant was freeze-dried in a -80℃ freeze dryer for 24h. The obtained freeze-dried sample was ground to prepare L-amino acid ligase (Lal) freeze-dried enzyme powder.

[0067] Example 4 Preparation of Lal from other sources

[0068] The vector construction and expression of L-amino acid ligase (Lal) from Pseudomonas syringae and the preparation of enzyme powder were the same as those in Example 1-3.

[0069] The vector construction and expression of L-amino acid ligase (Lal) from Ralstonia solanacearum and the preparation of enzyme powder were the same as those in Example 1-3.

[0070] The vector construction and expression of L-amino acid ligase (Lal) from Yersinia intermedia ATCC 29909 and the preparation of enzyme powder were the same as those in Example 1-3.

[0071] The vector construction and expression of L-amino acid ligase (Lal) from Streptomyces sp. SceAMP-e96 and the preparation of enzyme powder were the same as those in Example 1-3.

[0072] The vector construction and expression of L-amino acid ligase (Lal) from Bacillus velezensis and the preparation of enzyme powder were the same as those in Example 1-3.

[0073] The vector construction and expression of L-amino acid ligase (Lal) from Bacillus altitudinis and the preparation of enzyme powder were the same as those in Example 1-3.

[0074] The vector construction and expression of L-amino acid ligase (Lal) from Bacillus thuringiensis and the preparation of enzyme powder were the same as those in Example 1-3.

[0075] Example 5 Synthesis of L-valine-L-valine by Lal enzymes from different sources

[0076] 8 g L-valine (170 mmol) was added to 400 ml HEPES (50 mM) buffer solution, stirred at room temperature until completely dissolved, 18.7 g (85 mmol) ATP-2 Na salt was added, 0.38 g (10 mmol) MgCl2 was added, and the pH was adjusted to 8.0 with NaOH aqueous solution (5 M). After cooling to room temperature, 40 mg (0.5%) L-amino acid ligase (Lal) lyophilized enzyme powder was added to the reaction system, and NaOH aqueous solution (5 M) was added dropwise to control the reaction pH to be maintained at 7.5-8.0; 30 ° C, 400 rpm stirring for 20 hours, the reaction was completed, and the temperature was heated to 90 ° C, maintained for 10 minutes to inactivate the enzyme.

[0077] Derivatization

[0078] Take 100ul of the reaction solution, add 200ul of acetonitrile, add 100ul of boric acid (0.2M), add 100ul of Fmoc-Cl (850mmol), derivatize at 30℃ for 15min, add 100ul of 1-adamantanamine hydrochloride (0.1M, dissolved in acetonitrile: water = 1:1 solution), quench at room temperature for 10min, and centrifuge to take samples for detection.

[0079] Analysis and testing

[0080] Agilent 1290UHPLC was used to measure the product L-valine-L-valine to obtain the reaction conversion rate. The ultra-high performance liquid chromatography (UHPLC) was purchased from Agilent Technologies, and the UHPLC detection conditions were as follows: mobile phase A: water (containing 0.05% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.05% trifluoroacetic acid), temperature 35°C, flow rate 0.6 mL / min, and the chromatographic column was Agilent Poroshell 120EC-C18 (50 mm*2.1 mm, 1.9 μm).

[0081] The conversion rate calculation formula is as follows:

[0082] Conversion rate % = peak area [product / (raw material SM1+SM2)]*100%

[0083] The test results of the samples are as attached. Figure 1 As shown (taking the catalytic result of Lal from Bacillus subtilis as an example), where L-

[0084] The elution time of valine-L-valine is 1.61min, and the elution time of L-valine is 2.05min. The reaction results are shown in Table 1.

[0085] Table 1 Synthesis of L-valine by Lal enzymes from different sources - L-valine

[0086]

[0087] From the results, the NDT enzyme conversion rate from Bacillus subtilis was the highest at 95%.

[0088] Example 6 Synthesis of N-methyl-L-valine-L-valine by Lal enzymes from different sources

[0089] 4.6 g N-methyl-L-valine (85 mmol) and 4 g L-valine (85 mmol) were added to 400 ml HEPES (50 mM) buffer solution, stirred at room temperature until completely dissolved, 18.7 g ATP-2 Na salt (85 mmol) and 0.38 g MgCl2 (10 mmol) were added, and the pH was adjusted to 8.0 with a NaOH aqueous solution (5 M). After cooling to room temperature, 46 mg (0.53%) L-amino acid ligase (Lal) lyophilized enzyme powder was added to the reaction system, and a NaOH aqueous solution (5 M) was added dropwise to control the reaction pH to be maintained at 7.5-8.0; the reaction was stirred at 30° C. and 400 rpm for 20 h, and the reaction was completed, and the temperature was heated to 90° C. and maintained for 10 min to inactivate the enzyme.

[0090] The derivatization method of the sample is the same as that in Example 5.

[0091] The detection method of the sample is the same as that of Example 5. The detection results of the sample are shown in the attached Figure 2 (Taking the Lal catalytic results from Bacillus subtilis as an example), wherein the peak time of N-methyl-L-valine is 1.56 min, the peak time of N-methyl-L-valine-L-valine is 1.79 min, and the peak time of L-valine is 2.0 min. The reaction results are shown in Table 2.

[0092] Table 2 Synthesis of N-methyl-L-valine-L-valine by Lal enzymes from different sources

[0093]

[0094] From the results, the NDT enzyme conversion rate from Bacillus subtilis was the highest at 93%.

[0095] Example 7 Synthesis of N-ethyl-L-valine-L-valine by Lal enzymes from different sources

[0096] 4.9 g N-ethyl-L-valine (85 mmol) and 4 g L-valine (85 mmol) were added to 400 ml HEPES (50 mM) buffer solution, stirred at room temperature until completely dissolved, 18.7 g ATP-2 Na salt (85 mmol), 0.38 g MgCl2 (10 mmol) were added, and the pH was adjusted to 8.0 with NaOH aqueous solution (5 M). After cooling to room temperature, 49 mg (0.55%) L-amino acid ligase (Lal) lyophilized enzyme powder was added to the reaction system, and NaOH aqueous solution (5 M) was added dropwise to control the reaction pH to be maintained at 7.5-8.0; the reaction was stirred at 30° C. and 400 rpm for 20 h, and the reaction was completed, and the temperature was heated to 90° C. and maintained for 10 min to inactivate the enzyme.

[0097] The derivatization method of the sample is the same as that in Example 5.

[0098] The detection method of the sample is the same as that of Example 5. The detection results of the sample are shown in the attached Figure 3 As shown (taking the Lal catalytic result from Bacillus subtilis as an example), the peak time of N-ethyl-L-valine-L-valine is 1.64 min, the peak time of N-ethyl-L-valine is 1.85 min, and the peak time of L-valine is 2.05 min. The reaction results are shown in Table 3.

[0099] Table 3 Synthesis of N-ethyl-L-valine-L-valine by Lal enzymes from different sources

[0100]

[0101]

[0102] From the results, the NDT enzyme conversion rate from Bacillus subtilis was the highest at 89%.

[0103] Example 8 Synthesis of N-dimethyl-L-valine-L-valine by Lal enzymes from different sources

[0104] 4.9 g N, N-dimethyl-L-valine (85 mmol) and 4 g L-valine (85 mmol) were added to 400 ml HEPES (50 mM) buffer solution, stirred at room temperature until completely dissolved, 18.7 g ATP-2 Na salt (85 mmol) and 0.38 g MgCl2 (10 mmol) were added, and the pH was adjusted to 8.0 with a NaOH aqueous solution (5 M). After cooling to room temperature, 49 mg (0.55%) L-amino acid ligase (Lal) freeze-dried enzyme powder was added to the reaction system, and a NaOH aqueous solution (5 M) was added dropwise to control the reaction pH to be maintained at 7.5-8.0; the reaction was stirred at 30°C and 400 rpm for 20 hours, and the reaction was completed, and the temperature was heated to 90°C and maintained for 10 minutes to inactivate the enzyme.

[0105] The derivatization method of the sample is the same as that in Example 5.

[0106] The detection method of the sample is the same as that of Example 5. The detection results of the sample are shown in the attached Figure 4 As shown (taking the Lal catalytic result from Bacillus subtilis as an example), the elution time of N,N-dimethyl-L-valine-L-valine is 1.90 min, the elution time of L-valine is 2.06 min, and the elution time of N,N-dimethyl-L-valine is 2.25 min. The reaction results are shown in Table 4.

[0107] Table 4 Synthesis of N,N-dimethyl-L-valine-L-valine by Lal enzymes from different sources

[0108]

[0109] From the results, the NDT enzyme conversion rate from Bacillus subtilis was the highest at 82%.

[0110] Example 9 Synthesis of N-acetyl-L-valine-L-valine by Lal enzymes from different sources

[0111] 5.4 g N-acetyl-L-valine (85 mmol) and 4 g L-valine (85 mmol) were added to 400 ml HEPES (50 mM) buffer solution, stirred at room temperature until completely dissolved, 18.7 g ATP-2 Na salt (85 mmol) and 0.38 g MgCl2 (10 mmol) were added, and the pH was adjusted to 8.0 with a NaOH aqueous solution (5 M). After cooling to room temperature, 54 mg (0.57%) L-amino acid ligase (Lal) lyophilized enzyme powder was added to the reaction system, and a NaOH aqueous solution (5 M) was added dropwise to control the reaction pH to be maintained at 7.5-8.0; the reaction was stirred at 30° C. and 400 rpm for 20 h, and the reaction was completed. The temperature was heated to 90° C. and maintained for 10 min to inactivate the enzyme.

[0112] The derivatization method of the sample is the same as that in Example 5.

[0113] The detection method of the sample is the same as that of Example 5. The detection results of the sample are shown in the attached Figure 5 As shown (taking the Lal catalytic result from Bacillus subtilis as an example), the peak time of N-acetyl-L-valine-L-valine is 1.68 min, the peak time of N-acetyl-L-valine is 1.88 min, and the peak time of L-valine is 2.06 min. The reaction results are shown in Table 5.

[0114] Table 5 Synthesis of N-acetyl-L-valine-L-valine by Lal enzymes from different sources

[0115]

[0116] From the results, the NDT enzyme conversion rate from Bacillus subtilis was the highest at 86%.

[0117] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An L-amino acid ligase, characterized in that The amino acid sequence of the L-amino acid ligase comprises at least one of the following sequences: An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 2; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 3; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 4; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 5; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 6; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No:7; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No: 8; or An amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID No:

9.

2. The L-amino acid ligase according to claim 1, characterized in that The L-amino acid ligase is derived from Bacillus subtilis, Pseudomonas, Ralstonia solanacearum, Yersinia, Streptomyces, Bacillus velezensis, Bacillus thuringiensis or Bacillus thuringiensis.

3. The L-amino acid ligase according to claim 1, characterized in that The L-amino acid ligase is used for catalyzing the synthesis of L-valine dipeptide and its derivatives. The L-amino acid ligase is prepared by a genetic engineering strain constructed by in vitro recombination. The genetic engineering strain is Escherichia coli BL21, BL21 (DE3) and BL21Star (DE3).

4. A nucleic acid encoding an L-amino acid ligase, characterized in that The nucleic acid comprises a nucleotide sequence having at least 75% identity with the sequence shown in SEQ ID No:

1.

5. The nucleic acid encoding L-amino acid ligase according to claim 4, characterized in that The L-amino acid ligase encoded by the nucleic acid is derived from Bacillus subtilis.

6. A method for preparing an L-amino acid ligase, characterized in that: The preparation method is used to prepare the L-amino acid ligase according to claims 1-3, and the method comprises the following steps: 1) Construction of genetically engineered strains: The nucleotide sequence of L-amino acid ligase is digested and recombined into an expression vector, which is transformed into a host cell and plated on an LB solid agar plate to obtain an engineered strain containing the L-amino acid ligase gene; 2) selecting the positive clones of the genetically engineered strain obtained in step 1) and activating them in LB liquid medium, and then inoculating them in LB fermentation medium for culture, inducing expression, and obtaining an expression host containing the L-amino acid ligase; 3) The expression host obtained in step 2) is ultrasonically disrupted and centrifuged to obtain a supernatant, and the obtained supernatant is ultrafiltered, concentrated, and then freeze-dried to obtain a freeze-dried enzyme powder of L-amino acid ligase.

7. A method for catalytic synthesis of L-valine dipeptide and its derivatives, characterized in that: In an aqueous phase system, an L-valine analog reacts with L-valine under the action of an L-amino acid ligase, ATP and metal ions to generate an L-valine dipeptide and a derivative thereof, wherein the nucleotide sequence of the L-amino acid ligase is a nucleotide sequence having at least 75% identity with the sequence shown in SEQ ID No: 1, and the amino acid sequence of the L-amino acid ligase is an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID No:

2.

8. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The L-valine analog is selected from any one of L-valine, N-methyl-L-valine, N-ethyl-L-valine, N,N-dimethyl-L-valine or N-acetyl-L-valine.

9. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The pH of the aqueous phase system is 6.0-8.0, and the aqueous phase system is selected from any one of phosphate buffer, HEPES or Tris-HCl buffer.

10. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The mass concentrations of the L-valine analog and L-valine are both 0.1-100 mg / mL.

11. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The ATP is in the form of ATP or its salt ions, and the mass concentration of the ATP is 0.1-100 mg / mL.

12. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The metal ion is selected from any one of magnesium chloride, manganese chloride or cobalt chloride, and the mass concentration of the metal ion is 0.1-100 mg / mL.

13. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The L-amino acid ligase is L-amino acid ligase freeze-dried enzyme powder, and the dosage of the L-amino acid ligase freeze-dried enzyme powder is 0.1-10% of the weight of the L-valine analog.

14. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The reaction temperature is 25 to 40° C., and the reaction time is 12 to 24 hours.

15. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 7, characterized in that: The method comprises the following steps: S1, adding L-valine analogs and L-valine to an aqueous phase system, and then adding ATP and metal ions; S2, adjusting the pH of the reaction system to 6.0-8.0 using NaOH, adding L-amino acid ligase and maintaining the pH of the reaction system at 6.0-8.0; S3, raising the reaction temperature to 25-40°C and reacting for 12-24 hours; S4, raising the reaction temperature to 90°C to inactivate the enzyme; S5, adding a derivatization reagent to the reaction solution for derivatization; S6. Use UHPLC ultra-high performance liquid chromatography to analyze and detect the reaction conversion rate.

16. The method for catalytic synthesis of L-valine dipeptide and its derivatives according to claim 15, characterized in that: The derivatization reagent is Fmoc-Cl.

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