Alpha-amino acid ester acyltransferase mutant, amino acid sequence thereof and application

Abstract

The application discloses a kind of alpha-amino acid ester acyltransferase mutants, its amino acid sequence and application, the hydrolytic activity of the mutant is low, can greatly alleviate the degradation problem of product in later reaction period.It is used as the whole cell catalyst for synthesizing dipeptide, realizes clean, safe and efficient synthesis of propylvaline dipeptide, propylleucine dipeptide, leucine dipeptide and the like, effectively solves the problems of long reaction time and low substrate conversion rate in prior art, has very high application value and industrialization potential.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering applications, specifically involving a class of α-amino acid ester acyltransferase mutants, their amino acid sequences, and applications. Background Technology

[0002] L-Ala-Gln, also known as N(2)-L-alanyl-L-glutamine, has gradually replaced glutamine (L-Gln) as the main parenteral nutrition drug due to its high solubility, water solubility, and strong thermal stability. Currently, the mainstream production process of L-Ala-Gln is to achieve green and environmentally friendly production through efficient catalysis by biological enzymes. Researchers have obtained an amino acid ligase (Lal) from Bacillus subtilis and developed a patented technology for fermentation production of dipeptides. However, this catalytic synthesis of L-Ala-Gln requires exogenous ATP, resulting in a relatively high production cost and preventing its widespread use in the market (Tabata K, Hashimoto S. Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acidα-ligase. Applied and environmental microbiology, 2007, 73(20): 6378-6385.).

[0003] α-Amino acid ester acyltransferases can directly convert L-alanine methyl ester hydrochloride (L-AlaOMe) and L-glutamine into alanine-glutamine dipeptide. With its advantages of inexpensive and readily available raw materials, fewer reaction steps, mild reaction conditions, low environmental pollution, and few byproducts, it has become a novel method for synthesizing Ala-glutamine and has the potential for industrial production. Patent ZL201611254213.2 discloses a method for synthesizing alanine-glutamine dipeptide using heterologous expression of recombinant E. coli (Aet). This method can rapidly complete the catalytic reaction within 10 minutes, achieving a conversion rate as high as 73% in a 100mM substrate system. ZL201880014704.0 knocked out protease and / or peptidase genes in host cells and constructed a highly active α-amino acid ester acyltransferase, achieving extremely high conversion rates and yields within 5 minutes. However, reaching the catalytic peak too quickly leads to a product yield far lower than the theoretical yield in actual production, resulting in unsatisfactory economics. In 2005, the journal *J Biol Chem*, 2006, 281:5804-10, published "Acetobacter turbidans alpha-amino acid esterhydrolase: how a single mutation improves an antibiotic-producing enzyme," reported on the hydrolytic activity of the α-amino acid ester acyl hydrolase family on esters. In 2023, *Appl Microbiol Biotechnol*, 2023, 107:3523-3533, published "Multiplex gene knockout raises Ala-Gln production by *Escherichia coli* expressing amino acid ester acyltransferase," also demonstrated that knocking out the hydrolase in cells does not completely eliminate the dipeptide reverse reaction, indicating that α-amino acid ester acyltransferases themselves also possess hydrolytic activity, but their efficient synthetic capacity masks the hydrolytic effect.

[0004] Therefore, in practical production applications, an excessively fast catalytic rate is not a valuable process parameter. A bio-enzyme catalyst that can achieve a reasonable reaction rate and whose degradation is not significant after reaching its peak is urgently needed. Summary of the Invention

[0005] To address the aforementioned issues, this invention designs a class of α-amino acid ester acyltransferase mutants, their amino acid sequences, and their applications. These mutants reduce industrial costs and facilitate the advancement of industrialization.

[0006] One objective of this invention is to provide a class of α-amino acid ester acyltransferase mutants, wherein the α-amino acid ester acyltransferase mutants are selected from any mutation in the following group: G226S, G226A, G226T, G226D, and G226E; wherein the amino acid residue numbers are as shown in SEQ ID NO.2. Specifically, the amino acid residue numbers of the G226S transferase mutant are SEQ ID NO:5, the amino acid residue numbers of the G226A transferase mutant are SEQ ID NO:6, the amino acid residue numbers of the G226T transferase mutant are SEQ ID NO:7, the amino acid residue numbers of the G226D transferase mutant are SEQ ID NO:8, and the amino acid residue numbers of the G226E transferase mutant are SEQ ID NO:9.

[0007] Another object of the present invention is to provide a class of polynucleotides that encode genes for α-amino acid ester acyltransferase mutants as described in any of the preceding claims.

[0008] A type of recombinant expression vector, wherein the recombinant expression vector contains the polynucleotide gene fragment described above.

[0009] In a further preferred embodiment of the above-described technical solution, the recombinant expression vector is obtained by linking the gene fragment of the polynucleotide to an expression vector, wherein the expression vector preferably contains any one of the genes pET-29(a), pET28(a), and pET30(a).

[0010] A type of recombinant bacterial cell, wherein the recombinant bacterial cell contains the polynucleotides described above or expresses any of the α-amino acid ester acyltransferase mutants described above.

[0011] A method for preparing the α-amino acid ester acyltransferase mutant described in any of the preceding methods includes culturing the recombinant bacterial cells to express the α-amino acid ester acyltransferase mutant.

[0012] The above-described α-amino acid ester acyltransferase mutant, the polynucleotide, the recombinant expression vector, and the recombinant bacterial cells are used as synthetic dipeptide products.

[0013] For the technical solution described above, a further preferred embodiment is that the dipeptide product used in the application includes, but is not limited to, any one of: propionylglutathione dipeptide, ... and propionylglutathione dipeptide.

[0014] For the technical solution described above, a further preferred embodiment is that the reaction conditions in the application include: temperature 25-30℃, pH 8.0-9.0.

[0015] For the technical solution described above, a further preferred embodiment includes the step of preparing dipeptide products: in an environment of 25-30℃, a pH adjuster is added to a substrate solution of a certain concentration to make the pH of the reaction solution between 8.0 and 9.0, and the catalytic process is initiated using the α-amino acid ester acyltransferase mutant or recombinant bacterial cells described above.

[0016] For the technical solution described above, a further preferred embodiment is that the substrate solution contains at least one of alanine methyl ester hydrochloride, glycine methyl ester hydrochloride, leucine methyl ester hydrochloride, and isoleucine methyl ester hydrochloride, and at least one of glutamine, leucine, isoleucine, and tyrosine is added.

[0017] For the technical solution described above, a further preferred embodiment is that the concentration of the substrate solution is 100-500 mM.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention solves the bottleneck problems existing in the prior art and promotes its application in industrial production.

[0020] (1) This α-amino acid ester acyltransferase mutant exhibits low hydrolytic activity, which can greatly alleviate the degradation problem of products in the later stages of the reaction. The hydrolytic activity of propionyl dipeptide is reduced by 9.9%, that of propionyl dipeptide by 62%, and that of leucine dipeptide by 47%.

[0021] (2) Using α-amino acid ester acyltransferase mutants as whole-cell catalysts for the synthesis of dipeptides, we can achieve clean, safe and efficient synthesis of propionyl dipeptide, propionyl dipeptide, and glime dipeptide.

[0022] (3) As a whole-cell catalyst for the synthesis of dipeptides, the α-amino acid ester acyltransferase mutant can effectively solve the problem that the reaction rate is fast and the degradation is fast in the production of dipeptide products in the existing technology, resulting in a product yield far lower than the theoretical yield in actual production. Attached Figure Description

[0023] Figure 1 Hydrolytic activity of α-amino acid ester acyltransferases and their mutants;

[0024] Figure 2 Synthetic capacity of α-amino acid ester acyltransferases and their mutants;

[0025] Figure 3 α-Amino acid ester acyltransferases and their mutants are used to synthesize propionyl dipeptide;

[0026] Figure 4 α-Amino acid ester acyltransferases and their mutants are used to synthesize propionyl dipeptide;

[0027] Figure 5 α-Amino acid ester acyltransferases and their mutants are used to synthesize Brilliant dipeptide;

[0028] Figure 6 Comparison of the ability of α-amino acid ester acyltransferase saturated mutants to synthesize propionyl dipeptide. Detailed Implementation

[0029] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0030] In this invention, unless otherwise explicitly stated, percentages and contents are all by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available.

[0031] Example 1 Construction of mutants

[0032] Acquisition of mutants

[0033] 1. Using the SsAet base sequence (SEQ ID NO.1) in the pET29a-SsAet plasmid as a template, according to QuickMutation... TM The gene random mutation kit designs primers and performs random mutations. The primer sequences are as follows:

[0034] eqPCR-SsAet-F: tatcggatccATGAAAAATACAATTTCGTGCC; SEQ ID NO.3;

[0035] eqPCR-SsAet-R:gtggtggtggtggtgctcgagATCTTTGAGGACAGAAAATTCGATG;SEQ IDNO.4;

[0036] The amino acid sequence of the SsAet base sequence (SEQ ID NO.1) is shown in SEQ ID NO.2.

[0037] 2. Random Mutation PCR Reaction

[0038] Set up the random mutation PCR reaction system according to the table below:

[0039] Table 1

[0040]

[0041] Configure the PCR instrument according to the following parameters:

[0042] Table 2

[0043]

[0044]

[0045] 3. Transformation into competent cells

[0046] Take 10 μL of PCR product, detect with 1% agarose gel, and after observing the target band, add 1 μL of DMT enzyme to the remaining PCR product, mix well, and incubate for 1 h. Add 2-5 μL of digested product to 50 μL of DMT competent cells, mix well, incubate on ice for 30 min, then heat shock at 42℃ for 45 s, and immediately place on ice for 2-5 min. Then add 250 μL of LB medium, incubate at 37℃ for 1 h at 200 rpm, and take 100-200 μL of bacterial culture and incubate overnight on kanamycin-resistant plates. Several mutants were obtained, among which the mutants with prominent characteristics were named E. coli BL21-pET29(a)-Ssaet-G226S and E. coli BL21-pET29(a)-Ssaet-G226A, with the amino acid sequences of the proteins being SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0047] 4. Expression of wild-type strains and mutants

[0048] Recombinant *E. coli* strains E. coli BL21-pET29(a)-Ssaet, E. coli BL21-pET29(a)-Ssaet-G226S, and E. coli BL21-pET29(a)-Ssaet-G226A were cultured in LB medium overnight at 37°C and 200 rpm. They were then inoculated into fermentation medium at a 5% inoculum for scale-up culture. Cell OD was calculated. 600 =0.4~1.0, add 0.5mM IPTG, induce at low temperature overnight, and harvest whole cells by centrifugation as catalyst.

[0049] The seed culture medium and fermentation culture medium consist of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, sterilized at 121°C for 15 min, and then cooled before adding kanamycin at a final concentration of 75 mg / L.

[0050] Example 2: Comparison of hydrolysis activities

[0051] A 100 mM solution of propionylglutamate (PGD) was prepared, and the pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts from recombinant *E. coli* BL21-pET29(a)-Ssaet, *E. coli* BL21-pET29(a)-Ssaet-G226S, and *E. coli* BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken at 0, 10, and 20 min. The samples were centrifuged at 10,000 rpm for 60 s, and the precipitate was discarded. The supernatant was inactivated by heating at 99 °C for 3 min, and the samples were stored at 4 °C. The PGD hydrolyzed samples were brought to room temperature, and the hydrolysis rate was determined by high-performance liquid chromatography (HPLC).

[0052] Test results as follows Figure 1 As shown, the whole-cell enzyme catalyst of the mutant exhibits reduced hydrolytic activity towards the product. The hydrolysis rates of E. coli BL21-pET29(a)-Ssaet-G226S and E. coli BL21-pET29(a)-Ssaet-G226A are reduced by 9.9% and 5.4% respectively compared to E. coli BL21-pET29(a)-Ssaet. This indicates that the reverse reaction effect of the mutant is significantly reduced.

[0053] Example 3: Comparison of Synthesis Efficiency

[0054] A substrate solution was prepared with 100 mM alanine methyl ester hydrochloride and 150 mM glutamine. The pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts from recombinant *E. coli* BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken at 0, 10, and 20 min. The samples were centrifuged at 10,000 rpm for 60 s, the precipitate was discarded, and the supernatant was inactivated by heating at 99 °C for 3 min. The samples were stored at 4 °C. After the alanine-glutamyl dipeptide synthesis and hydrolysis samples were brought to room temperature, the synthesis yield was detected by high-performance liquid chromatography (HPLC).

[0055] Test results as follows Figure 2As shown, the catalytic efficiency of the mutant whole-cell enzyme catalyst was significantly improved. The conversion rates of E. coli BL21-pET29(a)-Ssaet-G226S (70.9±2.21%) and E. coli BL21-pET29(a)-Ssaet-G226A (79.23±1.65%) were 11.0% and 24.0% higher than those of E. coli BL21-pET29(a)-Ssaet (63.9±1.85%), respectively.

[0056] The synthesis-hydrolysis ratio can be defined as: (increase in the amount of glutathione during synthesis Δsynthesis + decrease in the amount of glutathione during hydrolysis Δhydrolysis) / decrease in the amount of glutathione during hydrolysis Δhydrolysis. The synthesis-hydrolysis ratios of glutathione for E. coli BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were 6.56, 10.09, and 15.67, respectively. It is evident that the synthesis-hydrolysis ratio of the mutant is significantly increased, indicating that the mutant has a stronger effect on the synthesis of glutathione.

[0057] Example 4: Hydrolytic activity of other dipeptides

[0058] Solutions of propranolol dipeptide and leucine dipeptide with a final concentration of 100 mM were prepared separately. The pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts of recombinant *E. coli* BL21-pET29(a)-Ssaet, *E. coli* BL21-pET29(a)-Ssaet-G226S, and *E. coli* BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken at 0, 10, and 20 min. The samples were centrifuged at 10,000 rpm for 60 s, the precipitate was discarded, and the supernatant was inactivated by heating at 99 °C for 3 min. The samples were stored at 4 °C. After the hydrolyzed samples were brought to room temperature, the hydrolysis rate was determined by high-performance liquid chromatography (HPLC).

[0059] The retention rates of the dipeptides are shown in Tables 1 and 2. It can be seen that the original strain exhibits a significant hydrolytic effect on propionyl dipeptide and leucine dipeptide. Without technical modifications to reduce the hydrolytic effect in the reaction, it is impossible to obtain a high concentration of leucine dipeptide.

[0060] Table 1. Retention concentration of propranolol dipeptide (mM)

[0061]

[0062]

[0063] Table 2. Retention concentration of Bright Bright Dipeptide (mM)

[0064]

[0065] Application Example 1: Synthesis of Propionylglutamic dipeptide

[0066] A substrate solution was prepared with 300 mM alanine methyl ester hydrochloride and 450 mM glutamine. The pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts from recombinant *E. coli* BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken after 30 min. The samples were centrifuged at 10,000 rpm for 60 s, and the precipitate was discarded. The supernatant was inactivated by heating at 99 °C for 3 min, and the samples were stored at 4 °C. The alanine-glutamine dipeptide synthesis samples were brought to room temperature, and the synthesis yield was detected by high-performance liquid chromatography (HPLC).

[0067] Test results as follows Figure 3 As shown, the conversion rates of E. coli BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were 66.33±4.16%, 73.8±1.71%, and 80.47±2.63%, respectively, and the product concentrations reached 43.22±2.71 g / L, 48.09±1.11 g / L, and 52.43±1.71 g / L, respectively, representing increases of 11.26% and 21.32%.

[0068] Application Example 2: Synthesis of Proleucine Dipeptide

[0069] A substrate solution was prepared with alanine methyl ester hydrochloride at a concentration of 300 mM and leucine at a concentration of 450 mM. The pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts from recombinant *E. coli* BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken after 30 min. The samples were centrifuged at 10,000 rpm for 60 s, and the precipitate was discarded. The supernatant was inactivated by heating at 99 °C for 3 min, and the samples were stored at 4 °C. The synthesized alanine-leucine dipeptide samples were brought to room temperature, and the synthesis yield was detected by high-performance liquid chromatography (HPLC).

[0070] Test results as follows Figure 4 As shown, the conversion rates of E. coli BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were 59.67±2.08%, 75.0±3.0%, and 64.67±3.51%, respectively, and the product concentrations reached 36.2±1.26 g / L, 45.51±1.82 g / L, and 39.24±2.13 g / L, respectively, representing increases of 25.7% and 8.4%.

[0071] Application Example 3: Synthesis of Bright Bright Dipeptide

[0072] A substrate solution was prepared with 200 mM leucine methyl ester hydrochloride and 300 mM leucine. The pH was adjusted to 8.5 using 6 mol / L NaOH solution. 5 mL of resuspended whole-cell enzyme catalysts from recombinant *E. coli* BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were added to achieve an OD of 2.0 in 100 mL of the reaction system. The reaction was carried out at 25 °C, and samples were taken after 30 min. The samples were centrifuged at 10,000 rpm for 60 s, and the precipitate was discarded. The supernatant was inactivated by heating at 99 °C for 3 min, and the samples were stored at 4 °C. The synthesized leucine dipeptide samples were brought to room temperature, and the synthesis yield was detected by high-performance liquid chromatography (HPLC).

[0073] Test results as follows Figure 5As shown, the conversion rates of E. coli BL21-pET29(a)-Ssaet, BL21-pET29(a)-Ssaet-G226S, and BL21-pET29(a)-Ssaet-G226A were 24.3±3.06%, 34.67±2.5%, and 40.7±2.8%, respectively, with product concentrations reaching 11.87±1.49 g / L, 16.94±1.22 g / L, and 19.89±1.37 g / L, representing increases of 42.7% and 67.5%, respectively. A saturation mutation of amino acid position 226 was also performed in the comparative example.

[0074] Since the mutation sites of the two better mutants are both at amino acid position 226, it is hypothesized that this amino acid has a special significance in the catalytic process. A saturation mutation was performed at this site, and the reaction was carried out according to the catalytic system of glutathione. The relative conversion rates are as follows: Figure 6 As shown, when amino acid 226 mutates to A, the relative concentration of glutathione reaches 125%; when mutated to S or T, the relative concentration of glutathione reaches about 110%; when mutated to D or E, the relative concentration of glutathione exceeds 10%; when mutated to M, C, V, K, F, W, P, L, Q, I, R, or H, the relative concentration of glutathione is very low, meaning that most mutations are non-positive mutations.

[0075] As can be seen from the results in the figure, only a few mutations have a positive effect, while most mutations have a very poor effect. Therefore, the positive mutations described in this application are not obvious.

[0076] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An α-amino acid ester acyltransferase mutant that increases the hydrolysis ratio in dipeptide synthesis, characterized in that: The amino acid sequence of the α-amino acid ester acyltransferase mutant is shown in SEQ ID NO.6, and the original amino acid residue numbers are those shown in SEQ ID NO.

2.

2. A polynucleotide, characterized in that: The polynucleotide encodes the gene for the α-amino acid ester acyltransferase mutant described in claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide as described in claim 2.

4. A recombinant bacterial cell, characterized in that: The recombinant bacterial cell contains the polynucleotide of claim 2 or expresses the α-amino acid ester acyltransferase mutant of claim 1.

5. A method for preparing the α-amino acid ester acyltransferase mutant of claim 1, comprising culturing the recombinant bacterial cells of claim 4 to express the α-amino acid ester acyltransferase mutant.

6. The application of the α-amino acid ester acyltransferase mutant as described in claim 1, or the polynucleotide as described in claim 2, or the recombinant expression vector as described in claim 3, or the gene recombinant bacterial cell as described in claim 4 in the preparation of synthetic pro-glutamyl dipeptide, pro-leucine dipeptide, and leucine dipeptide products.

7. The application according to claim 6, characterized in that: The reaction conditions for synthesizing the dipeptide product include: 25-30℃, pH 8.0-9.

0.

8. The application according to claim 6, characterized in that: The method for synthesizing the dipeptide product includes: initiating a catalytic process using the α-amino acid ester acyltransferase mutant of claim 1 or the recombinant bacterial cell of claim 4 under the conditions of substrate solution at 25-30°C and pH 8.0-9.

0.

9. The application according to claim 8, characterized in that: The substrate solution contains alanine methyl ester hydrochloride or leucine methyl ester hydrochloride, and glutamine or leucine is added.

Images