Alpha-amino acid ester acyltransferase mutants with high substrate conversion rates and use thereof
Patent Information
- Application Number
- CN202611432238.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-15
- Publication Date
- 2026-11-03
AI Technical Summary
The existing α-amino acid ester acyltransferases have low catalytic efficiency, resulting in low dipeptide yields, which limits the large-scale preparation and process economy of dipeptide products.
By mutating α-amino acid ester acyltransferases derived from Sphingosine monophosphate, mutation sites such as A208S, S307H, S100G, and Y185G were introduced to form mutants with high substrate conversion rates, thereby optimizing their catalytic performance.
It significantly improved the maximum conversion rate and reaction rate of dipeptides, increased product yield and synthesis rate, expanded substrate adaptability, and promoted the efficient and green biomanufacturing of dipeptide products.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a class of α-amino acid ester acyltransferase mutants with high substrate conversion rates and their applications, particularly in the biosynthesis of dipeptide products. Background Technology
[0002] Dipeptides are compounds composed of two amino acids linked by peptide bonds, and they have increasingly important applications in clinical nutrition, functional foods, and biomedicine. Ala-Gln (L-alanyl-L-glutamine), as a stabilized donor form of glutamine, overcomes the inherent defects of free glutamine, such as poor solubility, insufficient thermal stability, and spontaneous formation of pyroglutamic acid in aqueous solution. It has been widely used as a core component in parenteral nutrition formulations and serum-free cell culture media. Gly-Gln (L-glycyl-L-glutamine) shows unique advantages in maintaining the intestinal mucosal barrier and in high-density cell culture supplementation. Leucine dipeptide (L-leucyl-L-leucine, Leu-Leu), with its antioxidant, immunomodulatory, and muscle protein metabolism-promoting physiological functions, shows considerable market potential in sports nutrition and healthy aging product development.
[0003] Currently, the large-scale preparation of the aforementioned dipeptide products still relies primarily on chemical synthesis. Chemical synthesis methods typically depend on repeated amino protection and carboxyl activation steps, resulting in lengthy synthetic routes, low atom utilization, and the generation of large amounts of organic solvents and harmful byproducts, contradicting the development direction of green manufacturing. In recent years, enzymatic catalytic synthesis of dipeptides has attracted widespread attention from academia and industry. Among these, the method derived from *Sphingosine monophosphate* (…) Sphingobacterium siyangensis The α-amino acid ester acyltransferase (SsAet) has attracted much attention due to its unique catalytic properties. This enzyme can synthesize dipeptides in one step using unprotected amino acid esters as acyl donors and free amino acids as acyl acceptors, under mild conditions that do not depend on exogenous cofactors such as ATP. This simple and efficient catalytic mode makes it considered one of the preferred biocatalysts that may replace traditional chemical synthesis processes.
[0004] However, the catalytic efficiency of wild-type SsAet in practical applications has not yet reached the level expected for industrialization. A key issue is the limited maximum conversion rate of the enzyme-catalyzed synthesis reaction. Even with sufficient substrate, the final product concentration achievable in the reaction system is significantly lower than theoretically expected. This phenomenon stems from the inherent limitations of the enzyme's catalytic function, making it difficult to further improve the substrate-to-product conversion efficiency, thus restricting the improvement of product yield and overall process economy. This bottleneck also exists in the enzymatic synthesis of other high-value dipeptides such as glycine dipeptide and leucine dipeptide. Therefore, molecular modification of α-amino acid ester acyltransferases to break through their catalytic efficiency limits and significantly improve the maximum conversion rate is an urgent need for achieving efficient and green biomanufacturing of these dipeptide products. Summary of the Invention
[0005] To address the technical problem of low dipeptide yield due to the low conversion efficiency of existing α-amino acid ester acyltransferases, the present invention aims to provide a class of α-amino acid ester acyltransferase mutants with high substrate conversion efficiency and their applications, which significantly improves the conversion efficiency of α-amino acid ester acyltransferase mutants, with the aim of promoting the industrialization of enzymatic synthesis of dipeptides.
[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a class of α-amino acid ester acyltransferase mutants with high substrate conversion rates, wherein the mutants are derived from *Sphingosine monocytogenes* with amino acid sequences as shown in SEQ ID NO:1. Sphingobacterium siyangensis The mutant was obtained by mutating the wild-type α-amino acid ester acyltransferase SsAet, and the mutant contained at least one of the following mutation sites: A208S (alanine at position 208 is mutated to serine), S307H (serine at position 307 is mutated to histidine), S100G (serine at position 100 is mutated to glycine) and Y185G (tyrosine at position 185 is mutated to glycine).
[0007] Further, the α-amino acid ester acyltransferase mutant contains at least two mutation sites among A208S, S307H, S100G and Y185G; preferably, the α-amino acid ester acyltransferase mutant contains four mutation sites among A208S, S307H, S100G and Y185G.
[0008] Furthermore, the α-amino acid ester acyltransferase mutant can be obtained by modifying the gene encoding the wild-type α-amino acid ester acyltransferase SsAet with the amino acid sequence shown in SEQ ID NO:1 using conventional site-directed mutagenesis techniques in the art, or by directly obtaining the gene sequence encoding the mutant through whole-gene synthesis. The obtained gene is then ligated into an expression vector and introduced into host cells for expression to obtain the mutant enzyme.
[0009] Secondly, the present invention provides a class of coding genes capable of encoding the above-mentioned α-amino acid ester acyltransferase mutants; due to the degeneracy of codons, the nucleotide sequences of coding genes encoding the same amino acid sequence can exist in various forms, and these forms all fall within the protection scope of the present invention.
[0010] Thirdly, the present invention provides a type of recombinant expression vector containing the above-mentioned coding genes.
[0011] Based on the above technical solution, the recombinant expression vector further includes the pET-29a vector.
[0012] Fourthly, the present invention provides a type of recombinant bacteria carrying the above-mentioned recombinant expression vector and capable of expressing α-amino acid ester acyltransferase mutants.
[0013] Based on the above technical solution, the host of the recombinant bacteria is Escherichia coli, Pichia pastoris, or Saccharomyces cerevisiae.
[0014] Fifthly, the present invention provides a class of catalysts selected from one of the following forms: (1) Culture the above recombinant bacteria and isolate resting cells containing the α-amino acid ester acyltransferase mutant; (2) Frozen stem cells obtained by freeze-drying the resting cells; (3) The resting cells are disrupted to obtain a cell lysate containing the α-amino acid ester acyltransferase mutant; (4) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate; (5) The purified enzyme obtained after purification from the cell lysate; (6) Immobilized enzyme or immobilized cells obtained by immobilizing the purified enzyme or the resting cells.
[0015] In a sixth aspect, the present invention provides the application of the above-mentioned α-amino acid ester acyltransferase mutant, encoding gene, recombinant expression vector, recombinant bacteria or catalyst in the catalytic synthesis of dipeptides.
[0016] Based on the above technical solution, the dipeptide is further selected from at least one of Gly-Gln, Ala-Gln, Leu-Leu, Ala-Leu, Gly-Tyr, Ala-Tyr, Leu-Ile, Ile-Leu, and Ile-Ile.
[0017] Based on the above technical solution, the dipeptide is further defined as Gly-Gln, Ala-Gln, or Leu-Leu.
[0018] Based on the above technical solution, the synthesis reaction is further carried out in a buffer solution with a pH of 8.0 to 9.0 and a reaction temperature of 20 to 40°C.
[0019] Based on the above technical solutions, furthermore, the substrates for synthesizing Ala-Gln are alanine methyl ester and glutamine, with the concentration of alanine methyl ester being 10~1000 mM and the molar ratio of alanine methyl ester to glutamine being 1:1~1:3; the substrates for synthesizing Gly-Gln are glycine methyl ester and glutamine, with the concentration of glycine methyl ester being 10~1000 mM and the molar ratio of glycine methyl ester to glutamine being 1:1~1:3; and the substrates for synthesizing Leu-Leu are leucine methyl ester and leucine, with the concentration of leucine methyl ester being 10~1000 mM and the molar ratio of leucine methyl ester to leucine being 1:1~1:3.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Significantly improved maximum conversion rate: The α-amino acid ester acyltransferase mutant provided by the present invention significantly improves the maximum conversion rate of dipeptide products. Among them, the maximum conversion rate of the optimal tetra mutant of glutamic dipeptide is 16.0% higher than that of wild type, the maximum conversion rate of glutamic dipeptide is 20.2% higher than that of wild type, and the maximum conversion rate of glime dipeptide is 42.4% higher than that of wild type, which significantly improves the final yield of the product.
[0021] (2) Significantly improved reaction rate: The α-amino acid ester acyltransferase mutant provided by the present invention significantly improves the reaction rate of dipeptide products. Among them, the optimal tetra mutant has a 21.1% higher synthetic activity for propionyl dipeptide than the wild type, a 32.7% higher synthetic activity for glycine dipeptide than the wild type, and a 55.8% higher synthetic activity for glibenclamide dipeptide than the wild type, which effectively improves the reaction synthesis rate and reduces the preparation cost of whole-cell catalyst.
[0022] (3) Broad substrate spectrum: The α-amino acid ester acyltransferase mutant of the present invention can efficiently synthesize a variety of dipeptide products such as propionyl dipeptide, glycine dipeptide, and glime dipeptide, indicating that it has a wide substrate adaptability. Attached Figure Description
[0023] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.
[0024] Figure 1 A comparison of the maximum conversion rates of wild-type α-amino acid ester acyltransferases and various mutants for different dipeptides; Figure 2 A comparison diagram of the reaction process of wild-type α-amino acid ester acyltransferase and tetra mutant catalyzing the synthesis of Ala-Gln; Figure 3 A comparison diagram of the reaction process of wild-type α-amino acid ester acyltransferase and tetra mutant catalyzing the synthesis of Gly-Gln; Figure 4 This is a comparison diagram of the reaction process of wild-type α-amino acid ester acyltransferase and tetra-mutant catalyzing the synthesis of Leu-Leu.
[0025] Figure 5 The graph shows the catalytic performance of the four mutants under different pH conditions.
[0026] Figure 6 The figure shows the catalytic performance of the four mutants under different temperature conditions. Detailed Implementation
[0027] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.
[0028] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.
[0029] Example 1: Comparison of maximum conversion rates of mutants for the synthesis of different dipeptides This embodiment compares the catalytic performance of wild-type α-amino acid ester acyltransferase SsAet (amino acid sequence as shown in SEQ ID NO:1) and various mutants in Ala-Gln, Gly-Gln and Leu-Leu synthesis systems to obtain the maximum conversion rate of each mutant and screen out the superior mutants with rapid synthesis and significantly increased maximum conversion rate.
[0030] The reagents used in this embodiment include L-alanine methyl ester hydrochloride (AlaOMe, purity ≥98%), glycine methyl ester hydrochloride (GlyOMe, purity ≥98%), L-leucine methyl ester hydrochloride (LeuOMe, purity ≥98%), L-glutamine (Gln, purity ≥99%), L-leucine (Leu, purity ≥99%), L-alanyl-L-glutamine (Ala-Gln standard, purity ≥99%), glycyl-L-glutamine (Gly-Gln standard, purity ≥99%), L-leucyl-L-leucine (Leu-Leu standard, purity ≥99%), and phosphate buffered saline (PBS, pH 10). 8.5), and recombinant E. coli cells expressing wild-type α-amino acid ester acyltransferase SsAet, single-point mutants (A208S, S307H, S100G, Y185G) and quadruple mutants (containing all four mutation sites of A208S, S307H, S100G and Y185G).
[0031] The genes encoding the above-mentioned wild-type α-amino acid ester acyltransferases and each mutant were constructed into the pET-29a vector and transformed. E. coli BL21(DE3) competent cells; single colonies were picked and seeded in LB medium containing kanamycin and cultured at 37°C with shaking until OD. 620nm When the OD value is approximately 0.6-0.8, add IPTG to a final concentration of 0.6 mM and induce expression at 25°C for 12-16 h; collect the bacterial cells by centrifugation, resuspend in pre-cooled PBS buffer, and adjust the OD value. 620nm To the appropriate concentration (OD) 620nm =0.5), to obtain a whole-cell catalyst suspension.
[0032] Using AlaOMe / Gln, GlyOMe / Gln, and LeuOMe / Leu as substrate combinations (final concentrations of AlaOMe and GlyOMe were 100 mM, Gln was 200 mM, LeuOMe was 25 mM, and Leu was 50 mM), equal volumes of the above whole-cell catalyst suspensions (wild-type and each mutant) were added. The reactions were initiated at pH 8.5 and 30℃. Samples were taken at 60 min, centrifuged at 9000 rpm for 2 min to remove cells, and the supernatant was collected. The reaction was terminated by heating at 99℃ for 2 min to denature and precipitate residual enzyme proteins. The concentrations of the corresponding dipeptides in each reaction system were determined by HPLC using a HILIC-NH2 column and a mobile phase of 50 mM KH2PO4:acetonitrile = 35:65, detected under a 215 nm UV detector. The conversion rate was calculated based on the molar amount of the substrate amino acid methyl ester.
[0033] Experimental results are as follows Figure 1As shown, in the three synthetic systems of Ala-Gln, Gly-Gln, and Leu-Leu, the maximum transformation efficiency of each single-point mutant was higher than that of the wild type, with an increase ranging from 1.5% to 27.2%. Among them, the four mutants had the highest maximum transformation efficiency in all three systems: the Ala-Gln system achieved a maximum transformation efficiency of 75.4%, an increase of 16.0% compared to the wild type (maximum transformation efficiency of 65.0%); the Gly-Gln system achieved a maximum transformation efficiency of 70.7%, an increase of 20.2% compared to the wild type (maximum transformation efficiency of 58.8%); and the Leu-Leu system achieved a maximum transformation efficiency of 33.5%, an increase of 42.4% compared to the wild type (maximum transformation efficiency of 23.5%). These results indicate that the combination of the four beneficial mutation sites produced a synergistic effect, significantly exceeding the upper limit of the maximum transformation efficiency of wild-type SsAet.
[0034] Example 2: Comparison of reaction processes in the synthesis of Ala-Gln catalyzed by four mutants This embodiment confirms the advantage of the tetra mutant in terms of reaction rate by comparing the reaction process of the tetra mutant and the wild-type α-amino acid ester acyltransferase SsAet in the synthesis of Ala-Gln.
[0035] In this embodiment, the methods for culturing recombinant Escherichia coli cells, inducing expression, and preparing whole-cell catalyst suspension are the same as in Example 1; recombinant Escherichia coli whole cells expressing wild-type SsAet and quadruple mutants (containing all four mutation sites of A208S, S307H, S100G, and Y185G) are used as catalysts.
[0036] Using AlaOMe and Gln as substrates (final concentration of AlaOMe was 100 mM and final concentration of Gln was 200 mM), equal volumes of the above whole-cell catalyst suspension (wild-type SsAet and tetramutant) were added, and the reaction was started at pH 8.5 and 30℃. Samples were taken at 0, 5, 10, 15, 30 and 60 min, respectively. After the reaction was terminated according to the method in Example 1, the concentration of Ala-Gln was detected by HPLC, and the conversion rate was calculated based on the molar amount of alanine methyl ester. Conversion rate-time curves were plotted.
[0037] Experimental results are as follows Figure 2As shown in the figure, the conversion rate of the tetra-mutant was higher than that of the wild type at all sampling time points, and the product formation rate was significantly faster in the early stage of the reaction. The accumulation rate of Ala-Gln by the tetra-mutant within 5 min was 21.1% higher than that of the wild type. The conversion rate of the wild type tended to plateau after 30 min, reaching 65.0% at 60 min; while the tetra-mutant maintained a relatively fast growth rate from 30 min to 60 min, reaching 75.4% at 60 min. The above results indicate that the tetra-mutant not only has a higher maximum conversion rate in the synthesis of Ala-Gln, but also has a better reaction rate throughout the process than the wild type, which can effectively shorten the production cycle of Ala-Gln.
[0038] Example 3: Comparison of reaction processes in the synthesis of Gly-Gln catalyzed by four mutants This embodiment confirms the advantage of the tetra mutant in terms of reaction rate by comparing the reaction process of the tetra mutant and the wild-type α-amino acid ester acyltransferase SsAet in Gly-Gln synthesis.
[0039] In this embodiment, the methods for culturing recombinant E. coli cells, inducing expression, and preparing whole-cell catalyst suspension are the same as in Example 1. Recombinant E. coli whole cells expressing wild-type SsAet and quadruple mutants (containing all four mutation sites of A208S, S307H, S100G, and Y185G) were used as catalysts.
[0040] Using GlyOMe and Gln as substrates (final concentration of GlyOMe was 100 mM and final concentration of Gln was 200 mM), equal volumes of the above whole-cell catalyst suspension (wild-type SsAet and tetramutant) were added, and the reaction was initiated at pH 8.5 and 30℃. Samples were taken at 0, 5, 10, 15, 30, and 60 min, respectively. After the reaction was terminated according to the method in Example 1, the concentration of Gly-Gln was detected by HPLC, and the conversion rate was calculated based on the molar amount of glycine methyl ester. Conversion-time curves were plotted.
[0041] Experimental results are as follows Figure 3 As shown, the conversion rate of the tetra-mutant was higher than that of the wild type at all sampling time points, and the product formation rate was significantly faster in the initial stage of the reaction. The tetra-mutant accumulated Gly-Gln at a rate 32.7% higher than the wild type within 5 min; the conversion rate of the wild type plateaued after 30 min, reaching 58.8% at 60 min; while the tetra-mutant maintained a faster growth rate between 30 and 60 min, reaching 70.7% at 60 min. These results indicate that the tetra-mutant also exhibits a faster reaction rate and higher maximum conversion rate in Gly-Gln synthesis.
[0042] Example 4: Comparison of reaction processes for the four-mutant catalytic synthesis of Leu-Leu This embodiment confirms the advantage of the tetra mutant in terms of reaction rate by comparing the reaction process of the tetra mutant and the wild-type α-amino acid ester acyltransferase SsAet in Leu-Leu synthesis.
[0043] In this embodiment, the methods for culturing recombinant Escherichia coli cells, inducing expression, and preparing whole-cell catalyst suspension are the same as in Example 1; recombinant Escherichia coli whole cells expressing wild-type SsAet and quadruple mutants (containing all four mutation sites of A208S, S307H, S100G, and Y185G) are used as catalysts.
[0044] Using LeuOMe and Leu as substrates (LeuOMe final concentration of 25 mM and Leu final concentration of 50 mM), equal volumes of the above whole-cell catalyst suspension (wild-type SsAet and tetramutant) were added, and the reaction was started at pH 8.5 and 30℃. Samples were taken at 0, 5, 10, 15, 30 and 60 min, respectively. After the reaction was terminated according to the method in Example 1, the Leu-Leu concentration was detected by HPLC, and the conversion rate was calculated based on the molar amount of leucine methyl ester. Conversion rate-time curves were plotted.
[0045] Experimental results are as follows Figure 4 As shown, the conversion rate of the tetra-mutant was higher than that of the wild type at all sampling time points, and the product formation rate was significantly faster in the early stage of the reaction. The accumulation rate of Gly-Gln in the tetra-mutant was 55.8% higher than that of the wild type within 5 min. The conversion rate of the wild type tended to plateau after 15 min, reaching 23.5% at 60 min. However, the tetra-mutant maintained a stable growth rate from 15 min to 60 min, reaching 33.5% at 60 min. These results indicate that the tetra-mutant has a faster reaction rate and higher maximum conversion rate in the synthesis of Leu-Leu, which can effectively shorten the production cycle of Leu-Leu.
[0046] Example 5: Relative activity of tetramutants in catalyzing dipeptide synthesis at different temperatures and pH levels This embodiment aims to investigate the catalytic performance of the four mutants under different pH and temperature conditions, and to clarify their optimal reaction conditions and environmental adaptability.
[0047] Using AlaOMe / Gln, GlyOMe / Gln, and LeuOMe / Leu as substrate combinations (final concentrations of AlaOMe and GlyOMe were 100 mM, Gln was 200 mM, LeuOMe was 25 mM, and Leu was 50 mM), equal volumes of the above whole-cell catalyst suspensions (wild-type and each mutant) were added to initiate the reaction under different pH conditions (7.0, 7.5, 8.0, 8.5, 9.0, 9.5) and different temperatures (20℃, 25℃, 30℃, 35℃, 40℃). Samples were taken after 10 min, centrifuged at 9000 rpm for 2 min to remove cells, and the supernatant was collected. The reaction was terminated by heating at 99℃ for 2 min to denature and precipitate residual enzyme proteins. The concentrations of the corresponding dipeptides in each reaction system were determined by HPLC using a HILIC-NH2 column and a mobile phase of 50 mM KH2PO4:acetonitrile = 35:65, under a UV detector at a wavelength of 215 nm. Mutant activity was calculated based on the molar amount of the substrate amino acid methyl ester.
[0048] The catalytic performance results of the four mutants under different pH conditions are as follows: Figure 5 As shown, the optimal pH for both the tetramutant and the wild type in the three dipeptide synthesis systems was 8.5. The pH activity curves of both showed a similar trend, indicating that the introduction of the four mutation sites did not change the optimal pH of the enzyme. Within the pH range of 7.0 to 9.0, the conversion rate of the tetramutant in all three dipeptide systems was higher than that of the wild type. The catalytic performance of the tetramutant under different temperature conditions is shown in the following figures. Figure 6 As shown, the optimal temperature for both the tetra mutant and the wild type is 30℃, and their temperature-activity curves show the same trend, indicating that the mutation did not change the enzyme's optimal temperature. Within the range of 20 to 40℃, the conversion rate of the tetra mutant in all three dipeptide systems was higher than that of the wild type. These results demonstrate that the tetra mutant, while maintaining the same optimal pH and temperature as the wild type, exhibits higher catalytic efficiency over a wider pH and temperature range, demonstrating good environmental adaptability.
[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A class of α-amino acid ester acyltransferase mutants with high substrate conversion rates, characterized in that, The mutant is obtained by mutating the wild-type α-amino acid ester acyltransferase SsAet with the amino acid sequence shown in SEQ ID NO:
1. The mutant contains at least one of the following mutation sites: A208S (alanine at position 208 is mutated to serine), S307H (serine at position 307 is mutated to histidine), S100G (serine at position 100 is mutated to glycine), and Y185G (tyrosine at position 185 is mutated to glycine).
2. The α-amino acid ester acyltransferase mutant according to claim 1, characterized in that, The α-amino acid ester acyltransferase mutant contains at least two mutation sites among A208S, S307H, S100G and Y185G; preferably, the α-amino acid ester acyltransferase mutant contains four mutation sites among A208S, S307H, S100G and Y185G.
3. A class of coding genes encoding the α-amino acid ester acyltransferase mutants as described in claim 1 or 2.
4. A type of recombinant expression vector containing the coding gene as described in claim 3.
5. A type of recombinant bacteria carrying the recombinant expression vector of claim 4 and capable of expressing α-amino acid ester acyltransferase mutants.
6. The recombinant bacteria according to claim 5, characterized in that, The host of the recombinant bacteria is Escherichia coli, Pichia pastoris, or Saccharomyces cerevisiae.
7. A class of catalysts, characterized in that, The catalyst is selected from one of the following forms: (1) Culturing the recombinant bacteria of claim 5 or 6, and isolating resting cells containing the α-amino acid ester acyltransferase mutant; (2) Frozen stem cells obtained by freeze-drying the resting cells; (3) The resting cells are disrupted to obtain a cell lysate containing the α-amino acid ester acyltransferase mutant; (4) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate; (5) The purified enzyme obtained after purification from the cell lysate; (6) Immobilized enzyme or immobilized cells obtained by immobilizing the purified enzyme or the resting cells.
8. The application of the α-amino acid ester acyltransferase mutant of claim 1 or 2, the encoding gene of claim 3, the recombinant expression vector of claim 4, the recombinant bacteria of claim 5 or 6, or the catalyst of claim 7 in the catalytic synthesis of dipeptides, characterized in that, The dipeptide is at least one of Gly-Gln, Ala-Gln, Leu-Leu, Ala-Leu, Gly-Tyr, Ala-Tyr, Leu-Ile, Ile-Leu, and Ile-Ile; preferably, the dipeptide is Gly-Gln, Ala-Gln, or Leu-Leu.
9. The application according to claim 8, characterized in that, The synthesis of the dipeptide was carried out in a buffer solution with a pH of 8.0 to 9.0 at a temperature of 20 to 40°C.
10. The application according to claim 9, characterized in that, The substrates for synthesizing Ala-Gln dipeptide are alanine methyl ester and glutamine, with a concentration of alanine methyl ester ranging from 10 to 1000 mM and a molar ratio of alanine methyl ester to glutamine ranging from 1:1 to 1:3; the substrates for synthesizing Gly-Gln dipeptide are glycine methyl ester and glutamine, with a concentration of glycine methyl ester ranging from 10 to 1000 mM and a molar ratio of glycine methyl ester to glutamine ranging from 1:1 to 1:3; and the substrates for synthesizing Leu-Leu dipeptide are leucine methyl ester and leucine, with a concentration of leucine methyl ester ranging from 10 to 1000 mM and a molar ratio of leucine methyl ester to leucine ranging from 1:1 to 1:3.