An amino acid ester acyltransferase mutant, its encoding gene, and its application in the synthesis of alanine dipeptide.
By sequence mutation and expression of the amino acid ester acyltransferase CsAET, the substrate concentration tolerance and selectivity problems of amino acid ester acyltransferase-catalyzed synthesis of propionyl dipeptide in the prior art have been solved, realizing efficient and environmentally friendly synthesis of propionyl dipeptide.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2026-05-18
- Publication Date
- 2026-06-30
AI Technical Summary
Existing amino acid ester acyltransferase-catalyzed synthesis of propionyl dipeptide suffers from poor substrate concentration tolerance, poor selectivity at high substrate concentrations, and low target product concentration and yield.
By screening and modifying the amino acid ester acyltransferase CsAET, and by mutating the amino acid sequence, a mutant amino acid ester acyltransferase with high catalytic activity and selectivity was obtained. A recombinant expression vector was constructed and expressed in Escherichia coli, and a catalyst was prepared for the synthesis of propionyl dipeptide.
A high-yield and selective synthesis of propionylglutamate was achieved at high substrate concentrations. The process is simple, the reaction conditions are mild, and the environmental problems of chemical synthesis are avoided, showing good prospects for industrial application.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology and relates to an amino acid ester acyltransferase mutant that encodes a nucleic acid, a recombinant expression vector containing the nucleic acid sequence and a recombinant expression transformant, as well as a method for synthesizing propionyl dipeptide using the amino acid ester acyltransferase mutant or the recombinant expression transformant as a catalyst. Background Technology
[0002] L-Alanyl-L-Glutamine, also known as alanyl-L-glutamine dipeptide or glutamine, has the chemical formula C8H2O. 15 N3O4, with a relative molecular weight of 217.22 and CAS number 39537-23-0, is a white or off-white crystalline powder with a melting point of 215°C. It is odorless, hygroscopic, readily soluble in water, practically insoluble in methanol, and slightly soluble in glacial acetic acid.
[0003] L-Glutamine (Gln) is an amino acid widely distributed in the body, accounting for more than half of the total free amino acids. It is an important substance for regulating protein synthesis and breakdown, and an essential precursor for nucleic acid biosynthesis. It plays a crucial role in regulating the body's immune function and wound repair. However, L-glutamine has low solubility and poor stability in aqueous solutions. Furthermore, it easily generates toxic pyroglutamic acid upon heating, which significantly limits its clinical application as a drug due to its inability to withstand high-temperature sterilization. Propionylglutamate (PGD), on the other hand, is stable, can withstand high-temperature sterilization, has good water solubility, and a high rate of biodegradation in the body. It can rapidly hydrolyze to produce L-glutamine, exerting its effects. Its high bioavailability and wide range of applications compensate for the shortcomings of L-glutamine. PDD is widely used clinically for anti-fatigue, nutritional recovery, reducing immunosuppression in surgical and chemotherapy patients, and treating osteoporosis. It promotes immune function improvement and maintains the intestinal barrier, possessing significant medicinal value. As an amino acid-based parenteral nutrition agent, it is considered essential for patient care, similar to saline and glucose.
[0004] Currently, chemical synthesis methods are mainly used in industry to produce propionyl dipeptide, such as... D -Chloropropionyl chloride process, N -Hydroxysuccinimide method N- Carboxyl anhydride method. Chemical synthesis involves the protection and deprotection of active groups, involves multiple steps, and generates toxic substances during the chemical reaction, resulting in significant pollution emissions and serious environmental problems. Enzymatic synthesis offers advantages such as mild conditions and environmental friendliness. As the market for alpha-glutamyl dipeptide gradually expands, enzymatic synthesis is gaining increasing attention. Several enzymes capable of synthesizing alpha-glutamyl dipeptide have been reported, including amino acid ester acyltransferases (AET) and L-amino acid ligases (Lal). L-amino acid ligases can synthesize alpha-glutamyl dipeptide using L-alanine and L-glutamine as substrates under ATP-powered conditions. However, due to their low activity and ATP consumption, the production cost is high, making large-scale industrial application difficult. In contrast, amino acid ester acyltransferases have high activity, do not rely on ATP, and can synthesize alpha-glutamyl dipeptide with high substrate concentration and high conversion rate. These advantages give them great potential for industrial application.
[0005] The earliest discovery of an amino acid ester acyltransferase capable of catalyzing the synthesis of alanine dipeptide was made in 2005. Further research was conducted in strain [number missing] in 2011. Sphingobacterium siyangensis Highly active amino acid ester acyltransferase (SAET) was obtained from AJ2458 and successfully expressed heterologously. Biosci. Biotechnol. Biochem. (2011, 75, 2087). In recent years, multiple teams have focused on constructing expression strains, optimizing expression, and optimizing reaction systems for this enzyme. Using this enzyme to catalyze the conversion of 500 mM L-alanine methyl ester and 500 mM L-glutamine into alanine-glutamyl dipeptide, the product concentration was 321 mM, and the yield was 67%. Biosci. Biotechnol. Biochem. (2013, 77, 618). Under optimized conditions, this enzyme can catalyze the conversion of 600 mM L-alanine methyl ester and 480 mM L-glutamine into alanine-glutamyl dipeptide with a space-time yield of 117 g / (L*h) and a yield of 75% for L-glutamine, which is the highest reported level to date (Chinese Journal of Biotechnology, 2018, 1169). Nevertheless, this enzyme has poor substrate concentration tolerance, with a maximum substrate concentration of only 600 mM. The selectivity is poor at high substrate concentrations, which seriously limits the improvement of the concentration and yield of the target product, indicating significant room for improvement. Summary of the Invention
[0006] To address the problems of poor substrate concentration tolerance, poor selectivity at high substrate concentrations, and low target product concentration and yield in the enzymatic synthesis of propionylglutamic dipeptide catalyzed by amino acid ester acyltransferases, this invention provides an amino acid ester acyltransferase mutant with high catalytic activity, good selectivity, tolerance to high substrate concentrations, and a higher yield of propionylglutamic dipeptide formation compared to L-glutamine. The mutant encodes a nucleic acid, and the invention also includes a recombinant expression vector containing this nucleic acid sequence, a recombinant expression transformant, and a method for synthesizing propionylglutamic dipeptide using the amino acid ester acyltransferase mutant or the recombinant expression transformant as a catalyst.
[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions adopted in this invention: A mutant amino acid ester acyltransferase is provided, which is a mutant protein with amino acid ester acyltransferase activity obtained by substituting one or more amino acids into the amino acid sequence shown in SEQ ID No. 2.
[0008] This invention employs bioinformatics-assisted methods for enzyme screening. The amino acid sequence of SAET reported in the literature was used as a probe, and the NCBI protein BLAST function was used for searching. A phylogenetic tree was constructed, and different branches and different bacterial species from which the enzymes originated were used as selection criteria. Selected sequences were analyzed using expasy to predict their soluble expression. Finally, 20 candidate enzymes were selected for heterologous expression in *E. coli*. The activity of the enzymes was verified by catalyzing the synthesis of alanine-glutamyl dipeptide using the cell lysate of the recombinant expression transformants. The database number WP_313377242.1, with the amino acid sequence shown in SEQ ID No. 2, originated from *Li Changweni Chishuihe Bacterium* (…). Chishuiella amino acid ester acyltransferase (sp.) Cs AET exhibits higher activity in the synthesis of propionyl dipeptide than probe enzymes, and its selectivity in catalyzing the synthesis of propionyl dipeptide is also higher than that of probe enzymes. Cs The sequence identity between AET and SEAT was 65.8%.
[0009] Based on the discovery of new enzymes, amino acid ester acyltransferases Cs AET underwent semi-rational design modifications. Its structure was predicted and homology modeled. The Caver plugin in PyMol was used to predict its substrate channels, and amino acid residues on the substrate channel surface were mutated. After screening, a batch of amino acid ester acyltransferase mutants with reduced hydrolytic side reactions to the substrate alanine methyl ester and the product alanine dipeptide were obtained, and these were named... Cs AET mutant.
[0010] In one embodiment of the present invention, the amino acid sequence of the amino acid ester acyltransferase mutant is shown below.
[0011] (1) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (2) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (3) Replace the glycine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (4) Replace the glycine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (5) Replace the asparagine at position 71 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (6) Replace the asparagine at position 71 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (7) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (8) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (9) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (10) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (11) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (12) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (13) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (14) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (15) Replace the 90th position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (16) Replace the 90th position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (17) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with alanine; (18) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with isoleucine; (19) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine, and replace the asparagine at position 71 with alanine; (20) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 71 with isoleucine; (21) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the lysine at position 86 with alanine; (22) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and lysine at position 86 with isoleucine; (23) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the serine at position 87 with alanine; (24) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 87 with isoleucine; (25) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with alanine; (26) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with isoleucine; (27) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the tyrosine at position 158 with alanine; (28) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and tyrosine at position 158 with isoleucine; (29) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 90 with alanine; (30) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and replace the asparagine at position 90 with isoleucine.
[0012] Preferably, the amino acid sequence of the amino acid ester acyltransferase mutant is selected from (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (13), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (29), (30).
[0013] More preferably, the amino acid sequence of the amino acid ester acyltransferase mutant is selected from (1), (3), (7), (11), (25).
[0014] The second technical solution adopted in the invention: An isolated nucleic acid encoding an amino acid ester acyltransferase mutant as described in technical solution one, referred to simply as the amino acid ester acyltransferase mutant gene.
[0015] This invention provides a nucleic acid encoding an amino acid ester acyltransferase mutant, prepared using conventional methods in the art. The amino acid ester acyltransferase encoding the mutant was obtained through gene cloning technology. Cs The nucleic acid molecules of the AET mutant gene, in Forward primer 5'-ATCGGATCCATGAAAATACCCAAGATTAAAGTAAC-3' (SEQ ID NO.3) Reverse primer 5'-CGCAAGCTTGTTATTCAAAACCGG-3' (SEQ ID NO.4) The mutant DNA sequence encoding the amino acid ester acyltransferase CsAET obtained in Technical Scheme 1 was amplified using polymerase chain reaction (PCR) technology. PCR system (50 μL): 2× Prime Star Max 25 μL, template plasmid about 100 ng, forward and reverse primers 2 μL each, ddH2O to make up to 50 μL.
[0016] PCR reaction procedure: (1) 98°C pre-denaturation for 2 min; (2) 98°C denaturation for 15 s; (3) 60°C annealing for 15 s; (4) 72°C extension for 2 min; (5) Steps (2)-(4) are performed for a total of 32 cycles, and finally the extension is performed at 72°C for 10 min, and stored at 4°C.
[0017] The third technical solution adopted in this invention: A recombinant expression vector containing the amino acid ester acyltransferase mutant gene described in this invention is provided.
[0018] The amino acid ester acyltransferase mutant gene sequence of the present invention can be constructed by ligating it into various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, preferably a plasmid, more preferably plasmid pET-29a. The amino acid ester acyltransferase mutant gene can be operatively ligated downstream of a suitable regulatory sequence in the selected vector to achieve constitutive or inducible expression of the amino acid ester acyltransferase mutant.
[0019] Preferably, as an example, the recombinant expression vector of the present invention can be prepared by the following method: [The following is a description of a method for preparing the vector, which involves] an amino acid ester acyltransferase obtained by PCR amplification. Cs The gene sequence DNA fragment of the AET mutant was analyzed using restriction endonuclease. Bam HI and Hind III. Double digestion with restriction enzymes, and simultaneously digestion of the empty vector plasmid pET-29a with the same restriction endonuclease. Bam HI and Hind III. After double digestion with enzymes, the digested gene DNA fragments and pET-29a plasmid were recovered by gel extraction and ligated using T4 DNA ligase to obtain a recombinant expression plasmid containing the amino acid ester acyltransferase mutant gene.
[0020] The fourth technical solution adopted in this invention: A recombinant expression transformant comprising the amino acid ester acyltransferase mutant gene or its recombinant expression vector described in this invention is provided. The recombinant expression transformant can be prepared by transforming the recombinant expression vector described in this invention into conventional host cells. The host cells can be various conventional host cells in the art, provided that they can stably replicate spontaneously using the recombinant expression vector and that the amino acid ester acyltransferase gene they carry can be effectively expressed.
[0021] The preferred host cell of this invention is *Escherichia coli*, more preferably *Escherichia coli*. E. coli BL21(DE3).
[0022] The fifth technical solution adopted in this invention: An amino acid ester acyltransferase mutant catalyst is provided, comprising the following steps: culturing the recombinant expression transformant of the present invention, isolating resting cells containing the amino acid ester acyltransferase mutant; freeze-drying the cells to obtain frozen stem cells; lysing the resting cells to obtain cell lysate, and freeze-drying the cell lysate to obtain lyophilized enzyme powder.
[0023] The culture medium used to culture the recombinant expression transformant can be selected from conventional culture media in the art, provided that it enables the transformant to grow and produce the amino acid ester acyltransferase mutant of the present invention. Specific procedures for culturing the transformant can be performed according to conventional procedures in the art.
[0024] The plasmid carrying the amino acid ester acyltransferase encoding sequence was transformed into... Escherichia coli BL21(DE3) competent cells. The transformed strain was plated on LB solid medium containing 50 μg / mL kanamycin and incubated at 37°C for 12–14 h. Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin in test tubes and cultured with shaking at 37°C and 200 rpm for 12–14 h. 50 μL of the overnight culture was then inoculated into TB liquid shake flask medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600 Approximately 0.6–0.8 (about 3 h) of isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and expression was induced for 16–20 h at 16°C and 200 rpm. The bacterial culture was collected and incubated at 4°C and 8000× g Centrifuge for 10 min under the specified conditions to collect the resting cells; freeze-dry the bacterial pellet to obtain frozen stem cells. Resuspend the bacterial cells in pre-cooled PBS buffer (pH 8.5), and then use an ultrasonic homogenizer to homogenize the cells in an ice-water bath (30% power, 4 s sonication, 6 s interval, total time 15 min) to obtain crude enzyme solution. Freeze-dry the crude enzyme solution to obtain lyophilized enzyme powder.
[0025] The amino acid ester acyltransferase mutant expressed by plasmid pET-29a in this invention possesses a histidine tag (His-Tag), allowing for protein purification using a Ni NTABeads6FF nickel column. After obtaining the crude enzyme solution, it was centrifuged at 4°C and 12000 rpm for 50 min. The supernatant was collected, filtered through a 0.46 μm filter, and loaded onto the nickel column. Solutions A, B, and C were prepared using PBS, where solution A contained 10 mM imidazole, solution B contained 500 mM imidazole, and solution C contained 1 mM DTT and 5% glycerol. After pretreatment of the affinity column, the filtered supernatant was added, followed by elution with 10 column volumes of solution A, 95% solution A + 5% solution B, and 70% solution A + 30% solution B in that order. The eluent of 70% solution A + 30% solution B was collected, and imidazole was removed by replacement with solution C using a 30 kDa ultrafiltration tube. The solution was then concentrated to obtain a pure enzyme solution. The purification of the protein was analyzed by SDS-PAGE, and the protein concentration was determined using Nanodrop. After aliquoting, glycerol was added to a final concentration of 30%, and the solution was stored at -80°C for later use.
[0026] The sixth technical solution adopted in this invention: The present invention provides an amino acid ester acyltransferase. Cs Application of AET and its mutants in the catalytic condensation of L-alanine methyl ester and L-glutamine to prepare propionyl-glutamyl dipeptide.
[0027] The application described herein is the use of the amino acid ester acyltransferase described in this invention. Cs AET and its mutant catalysts are added to a buffer solution containing L-alanine methyl ester and L-glutamine to catalyze the synthesis of alanine-glutamyl dipeptide from L-alanine methyl ester and L-glutamine. The method is characterized by a reaction temperature of 20-40°C, a reaction pH of 7.0-9.0, and a concentration of both L-alanine methyl ester and L-glutamine at 100-1000 mM.
[0028] Intermittent sampling was performed during the reaction, and analysis was conducted using liquid chromatography. A chiral crown ether column, CROWNPAK® CR(+) 4.0 mm × 150 mm, with a packing size of 5 μm, was used as the liquid chromatography column. The mobile phase was perchloric acid (HCO4) at pH 1.0, the flow rate was set to 0.3 mL / min, the column oven temperature was set to 30°C, and detection was performed at a wavelength of 210 nm.
[0029] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The amino acid acyltransferase mutant described in this invention has significant advantages such as high activity, good selectivity, and tolerance to high substrate concentrations. Using this enzyme to catalyze the condensation of L-alanine methyl ester and L-glutamine via acyltransferase reaction to prepare propionyl-glutamyl dipeptide is a simple process with mild reaction conditions, high space-time yield, and is environmentally friendly. It avoids the group protection and deprotection processes and complex reaction conditions required in conventional chemical synthesis of propionyl-glutamyl dipeptide, and has promising application prospects in the industrial production of propionyl-glutamyl dipeptide. Detailed Implementation
[0030] The present invention will now be described in detail with reference to specific embodiments.
[0031] Example 1: Screening of amino acid ester acyltransferases.
[0032] Using the amino acid sequences of SAET and EAET reported in the literature as probes, the NCBI protein BLAST function was used for searching. For the query results of the two amino acid sequences, a sequence similarity range of 20%-70% was set, displaying 5000 query sequences. After removing redundant sequences, the obtained sequences were subjected to sequence similarity network analysis using the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EFT). A subset of sequences was selected from each cluster, ultimately yielding 248 sequences. Sequence alignment was performed using mega11 software, and a phylogenetic tree was constructed using the maximum likelihood method. Different evolutionary branches and different bacterial species were selected as selection criteria. Selected sequences were analyzed using expasy to predict soluble expression, ultimately identifying 20 candidate enzymes. The corresponding amino acid ester acyltransferase recombinant plasmids were obtained through PCR or artificial synthesis and transformed into *E. coli* to obtain the corresponding recombinant strains. The strains were inoculated into test tubes containing 4 mL of culture medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 1 mL of the bacterial culture was transferred to a shake flask containing 100 mL of TB culture medium for induction culture. After 24 h, the bacterial cells were collected by centrifugation. The cells were resuspended in 10 mL of PBS buffer (100 mM, pH 8.5) and sonicated in an ice-water bath at 30% power for 15 min to obtain the lysate.
[0033] The activity of the crude enzyme solution in synthesizing alanine-glutamyl dipeptide was verified by biocatalytic reaction. The total reaction volume was 1 mL, containing 100 mM L-alanine methyl ester, 100 mM L-glutamine, 100 mM PBS (pH 8.5), and an appropriate amount of crude enzyme solution. The reaction was carried out at 25°C and 1000 rpm for 10 min with shaking. After the reaction was completed, 10 μL of the reaction solution was mixed with 990 μL of perchloric acid to terminate the reaction. The reaction solution was then subjected to a 13000 × 10⁻⁶ reaction. g After centrifugation for 2 min, the sample was filtered through a mixed cellulose membrane and analyzed by high-performance liquid chromatography (HPLC). The results were used to determine whether the screened new enzyme had the activity to synthesize glutamic-peptide. The results are shown in Table 1.
[0034] Table 1 Comparison of propionyl dipeptide activity among different strains SAET and EAET are probe sequences; "++" indicates that the synthetic activity is higher than that of the probe; "+" indicates that the synthetic activity is lower than that of the probe, but there is synthetic activity for glutathione; "-" indicates that no synthetic activity for glutathione was detected.
[0035] Example 2 Recombinant amino acid ester acyltransferase Cs Preparation of AET The amino acid ester acyltransferase with NCBI number WP_313377242.1 as described in Example 1 Cs The amino acid sequence of AET is shown in SEQ ID No. 2 of the sequence listing. Using the forward primer 5'-ATCGGATCCATGAAAATACCCAAGATTAAAGTAAC-3' and the reverse primer 5'-CGCAAGCTTGTTATTCAAAACCGG-3', the coding gene of the sequence shown in SEQ ID No. 1 was amplified using polymerase chain reaction (PCR). The amplified coding DNA fragment was then processed with restriction endonucleases. Bam HI and Hind III double digestion, and then subjected to the same process Bam HI and Hind The empty vector plasmid pET-29a, which was digested with enzyme III, was ligated to obtain the recombinant plasmid pET-29a- Cs AET.
[0036] The obtained recombinant plasmid was transformed into *E. coli* to obtain the corresponding recombinant strain. The strain was inoculated into a test tube containing 4 mL of culture medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 1 mL of the bacterial culture was transferred to a shake flask containing 100 mL of TB culture medium and induced for 24 h. The bacterial cells were collected by centrifugation using a large centrifuge. The bacterial cells were resuspended in 10 mL of PBS buffer (100 mM, pH 8.5) and sonicated at 30% power for 15 min in an ice-water bath to obtain the lysate. The lysate was centrifuged at 4°C and 12000 rpm for 50 min, and the supernatant was collected and filtered through a 0.46 μm filter membrane. The crude enzyme solution, after centrifugation and filtration, was loaded onto a nickel column washed with five column volumes of deionized water. Elution was then performed sequentially with 10 column volumes of solution A, 95% solution A + 5% solution B, and 70% solution A + 30% solution B. The eluent of 70% solution A + 30% solution B was collected, and imidazole was removed using a 30 kDa ultrafiltration tube and solution C. The solution was then concentrated to obtain a pure enzyme solution, aliquoted, and glycerol was added to a final concentration of 30%. The solution was stored at -80°C for later use. Analysis showed... Cs The synthetic activity of the purified AET enzyme is 450 U / mg.
[0037] Example 3 Recombinant amino acid ester acyltransferase Cs Molecular modification of AET amino acid ester acyltransferase Cs The structure of AET was predicted and homology modeled. The substrate channel was predicted using the caver plugin in PyMol, and amino acid residues on the substrate channel surface were mutated. Mutations were performed on some amino acid residues constituting the substrate channel using alanine and isoleucine scans. After obtaining the mutants, the culture, activity assay, and detection methods were the same as described in Example 1. The crude enzyme synthesis activity of 16 mutants was measured, and it was found that they were similar to the parent enzyme. Cs Compared to the crude AET enzyme solution, some variants showed increased activity, as shown in Table 2. The sequence numbers in Table 2 correspond to a series of sequences listed later in the table. In the activity column, the activity of the variants was increased compared to the parent strain. Cs Compared to AET, one plus sign "+" represents a 1-2 times increase in vitality, two plus signs "++" represent a more than two times increase in vitality, and a minus sign "-" represents no change or no increase in vitality.
[0038] Table 2. List of amino acid ester acyltransferase mutant sequences and corresponding improvements in the synthetic activity of glutamic-glucan dipeptide. The amino acid sequences of the CsAET mutants corresponding to the labels in the table are as follows: (1) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0039] (2) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0040] (3) Replace the 18th glycine in the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0041] (4) Replace the 18th glycine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0042] (5) Replace the 71st position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0043] (6) Replace the 71st position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0044] (7) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0045] (8) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0046] (9) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0047] (10) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0048] (11) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0049] (12) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0050] (13) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0051] (14) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0052] (15) Replace the 90th position of the amino acid sequence shown in SEQ ID No. 2 with alanine.
[0053] (16) Replace the 90th position of the amino acid sequence shown in SEQ ID No. 2 with isoleucine.
[0054] (17) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with alanine; (18) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with isoleucine; (19) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine, and replace the asparagine at position 71 with alanine; (20) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 71 with isoleucine; (21) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the lysine at position 86 with alanine; (22) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and lysine at position 86 with isoleucine; (23) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the serine at position 87 with alanine; (24) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 87 with isoleucine; (25) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with alanine; (26) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with isoleucine; (27) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the tyrosine at position 158 with alanine; (28) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and tyrosine at position 158 with isoleucine; (29) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 90 with alanine; (30) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and replace the asparagine at position 90 with isoleucine.
[0055] Example 4 Amino acid ester acyltransferase mutant Cs AET M25 Preparation Extract the recombinant plasmid pET-29a- obtained as in Example 3. Cs AET M25 Transform it into E. coli E. coli In BL21, the strain was inoculated into a test tube containing 4 mL of culture medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 1 mL of the bacterial culture was transferred to a shake flask containing 100 mL of TB culture medium for induction culture. After 24 h, the bacterial cells were collected by centrifugation. The collected bacterial cells were pre-frozen at -80°C for 12–16 h and then freeze-dried at -55°C and 100 Pa for 48 h to obtain frozen stem cells.
[0056] 1 g of wet bacterial cells were resuspended in 10 mL of PBS buffer (100 mM, pH 8.5) and sonicated at 30% power for 15 min in an ice-water bath to obtain the lysate. The lysate was pre-frozen at -20°C for 12-16 h and then freeze-dried at -55°C and 100 Pa for 48 h to obtain the lyophilized enzyme powder.
[0057] The crude enzyme solution was centrifuged at 4 °C and 12,000 rpm for 50 min. The supernatant was collected, filtered through a 0.46 μm filter membrane, and loaded onto a nickel column rinsed with five column volumes of deionized water. Elution was then performed in the following order: 10 column volumes of solution A, 95% solution A + 5% solution B, and 70% solution A + 30% solution B. The eluent of 70% solution A + 30% solution B was collected, and imidazole was removed by replacement with a 30 kDa ultrafiltration tube and solution C. The solution was then concentrated to obtain a pure enzyme solution. After aliquoting, glycerol was added to a final concentration of 30% and stored at -80 °C for later use.
[0058] Example 5: Combination mutants of amino acid ester acyltransferases Cs AET M25 Preparation Extract the recombinant plasmid pET-29a- obtained as in Example 3. Cs AET M25 Using the forward primer 5'-AGCTATGCCGGCTTTTATAGCACCGTCAGC and the reverse primer 5'-AAAGCCGGCATAGCTAATGCCGTAGATCCC, polymerase chain reaction (PCR) was employed to target pET-29a- Cs AET M25 The gene encoding the sequence was amplified using... Dpn I. Enzyme digestion of gene amplification template pET-29a- Cs AET M25 , obtained pET-29a- Cs AET M25 .
[0059] The obtained plasmid was transformed into *E. coli* to obtain the corresponding bacterial strain. The strain was inoculated into a test tube containing 4 mL of culture medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 1 mL of the bacterial culture was transferred to a shake flask containing 100 mL of TB culture medium and induced for 24 h. The bacterial cells were collected by centrifugation using a large centrifuge. The bacterial cells were resuspended in 10 mL of PBS buffer (100 mM, pH 8.5) and sonicated at 30% power for 15 min in an ice-water bath to obtain the lysate. The lysate was centrifuged at 4°C and 12000 rpm for 50 min, and the supernatant was collected and filtered through a 0.46 μm filter membrane. The crude enzyme solution, after centrifugation and filtration, was loaded onto a nickel column washed with five column volumes of deionized water. Elution was then performed in the following order: 10 column volumes of solution A, 95% solution A + 5% solution B, and 70% solution A + 30% solution B. The eluent of 70% solution A + 30% solution B was collected, and imidazole was removed by replacement with a 30 kDa ultrafiltration tube and solution C. The solution was then concentrated to obtain a pure enzyme solution. After aliquoting, glycerol was added to a final concentration of 30% and stored at -80°C for later use.
[0060] Example 6 Temperature effect on amino acid ester acyltransferase Cs AET M25 The impact of vitality.
[0061] The activity of this enzyme in synthesizing alanine-glutamyl dipeptide was determined at different temperatures (20-40°C). The total reaction volume was 1 mL, containing 100 mM L-alanine methyl ester, 100 mM L-glutamine, 100 mM PBS (pH 8.5), and an appropriate amount of purified enzyme solution. The reaction was shaken for 10 min, and samples were taken for HPLC analysis. The results are shown in Table 3. The enzyme exhibited the highest activity at 30°C; this temperature was defined as 100% of the enzyme activity, and the relative activities at other temperatures were calculated. Before 30°C, the enzyme activity increased with increasing temperature; above 30°C, the enzyme activity decreased rapidly.
[0062] Table 3 Amino acid ester acyltransferases Cs AET M25 Activity at different temperatures Example 7 pH-dependent amino acid ester acyltransferase Cs AET M25 Effect of activity The activity of this enzyme in synthesizing alanine-glutamyl dipeptide was determined at 25°C in different pH buffers. The total reaction volume was 1 mL, containing 100 mM L-alanine methyl ester, 100 mM L-glutamine, PBS buffer (pH 6.0–9.0), and an appropriate amount of purified enzyme solution. The reaction mixture was shaken for 10 min, and samples were taken for HPLC analysis. The results are shown in Table 4. The enzyme exhibited the highest relative activity in PBS buffer at pH 8.5, defined as 100%. The relative activities at other pH values were calculated.
[0063] Table 4. Amino acid ester acyltransferase CsAET M25 Activity at different pH levels Example 8. CsAET, an amino acid ester acyltransferase, under different substrate concentrations M25 Catalytic synthesis of propionylglutamic dipeptide The reaction was carried out in a 50 mL jacketed glass reactor. A certain amount of L-glutamine and L-alanine methyl ester were weighed and added to the reactor, followed by deionized water to a final volume of 40 mL. The mixture was stirred to ensure complete dissolution of the substrate. 400 U of frozen stem cells as described in Example 4 were added, and the reaction was initiated at 20°C. 6M NaOH was added to the system using a titrator to maintain a constant pH of 8.5. Samples were taken periodically and analyzed using HPLC. The highest concentration of L-glutamine dipeptide and the maximum yield of glutamine were recorded for each batch of reaction.
[0064] Table 5 Amino acid ester acyltransferases Cs AET M25 Highest concentration and yield of catalytic synthesis of propionylglutamic dipeptide Example 9 Amino acid ester acyltransferase Cs AET M25 Catalytic synthesis of propionylglutamic dipeptide The reaction was carried out in a 50 mL jacketed glass reactor. 30 mmol L-glutamine and 10 mmol L-alanine methyl ester were weighed and added to the reactor, followed by deionized water to a final volume of 40 mL. The mixture was stirred to ensure complete dissolution of the substrate. 800 U of frozen stem cells as described in Example 4 were added, and the reaction was initiated at 20°C. 6 M NaOH was added to the system using a titrator to maintain a constant pH of 8.5. 10 mmol of L-alanine methyl ester was added at 10, 30, and 60 min after the start of the reaction. After 2 h of reaction, the concentration of L-glutamine dipeptide was 557 mM, and the maximum yield of glutamine reached 92.7%.
[0065] The sequence information involved in this invention is as follows: The sequences involved in this invention are as follows: SEQ ID No. 1 SEQ ID No. 2 MKIPKIKVTAIISFSLLGSIAFSQDAKADSIYVREHYDKVEQLIPMRDGTKLFTSIYIPKDKSHSYPVLLNRTPYTVAPYGDDYKKSLGNFPAEMREGFIFVYQDVRGKWMSEGTFEDVRPTNKSKNKKAFDESTDTYDTLEWLSKNLKNYNKK AGIYGISYPGFYSTVSLINSHPTLKAVSPQAPVTDWFIGDDFHRNGVLYLNDSFRFMSTFGVNRPHPITPDQGPKPINYPIKDIYRFNLEAGSVKELKDKYFQNNIKFYNDIFAHPNYDEFWQERNPLVNLTDVKPAVMTVAGFFDAEDAWGAFA TYKAIEKQNPKANNVLVAGPWFHGGWVRSKGDTFGDMQFDNPTGEYYQQNIELPFFNYYLKGKGDYKPTEAHIFISGSNEWKQFESWPPKNTTSKKMYLQENGKIAFDQPTAPNSFDEYVADPNNPVPFQGGVLETRSREYMVDDQRFASTRPD VMVYQTDVLDSDITLTGPIINHLFVSSTGTDADYLVKLIDVYPEDTPRFNGKLMGGYQNLIRGEIMRGKYRNSYEKPEALIPNEKTSVTYSMPDVGHTFKKGHRIMIQVQNSWYPLADRNPQQFMNVYEATAKDFLKQTQRIYHDSYIEVPVLNN SEQ ID NO.3 ATCGGATCCATGAAATACCCAAGATTAAAGTAAC SEQ ID NO.4 CGCAAGCTTGTTATTCAAAACCGG The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. An amino acid ester acyltransferase mutant, characterized in that, The amino acid sequence of the amino acid ester acyltransferase mutant is shown below: (1) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (2) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (3) Replace the glycine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (4) Replace the glycine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (5) Replace the asparagine at position 71 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (6) Replace the asparagine at position 71 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (7) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (8) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (9) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (10) Replace the 87th serine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (11) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (12) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (13) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (14) Replace the tyrosine at position 158 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (15) Replace the 90th position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (16) Replace the 90th position of asparagine in the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (17) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with alanine; (18) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and glycine at position 18 with isoleucine; (19) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine, and replace the asparagine at position 71 with alanine; (20) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 71 with isoleucine; (21) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the lysine at position 86 with alanine; (22) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and lysine at position 86 with isoleucine; (23) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the serine at position 87 with alanine; (24) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 87 with isoleucine; (25) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with alanine; (26) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and serine at position 19 with isoleucine; (27) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the tyrosine at position 158 with alanine; (28) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and tyrosine at position 158 with isoleucine; (29) Replace proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and asparagine at position 90 with alanine; (30) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and replace the asparagine at position 90 with isoleucine.
2. The amino acid ester acyltransferase mutant according to claim 1, characterized in that, The amino acid sequence of the amino acid ester acyltransferase mutant is shown below: (1) Replace the 19th serine in the amino acid sequence shown in SEQ ID No. 2 with alanine; (3) Replace the glycine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (7) Replace the lysine at position 86 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (11) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (25) Replace the proline at position 163 of the amino acid sequence shown in SEQ ID No. 2 with alanine and the serine at position 19 with alanine.
3. An isolated nucleic acid, characterized in that, The nucleic acid encodes the amino acid ester acyltransferase mutant as described in claim 1 or 2.
4. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 3.
5. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector as described in claim 4.
6. A mutant catalyst of an amino acid ester acyltransferase, characterized in that, It is any of the following forms: (1) Cultivate the recombinant expression transformant as described in claim 5, and isolate resting cells containing the amino acid ester acyltransferase mutant as described in claim 1; (2) Frozen stem cells obtained by freeze-drying resting cells containing amino acid ester acyltransferase mutants as described in form (1); (3) Disrupt the resting cells described in form (1) to obtain a cell lysate containing the amino acid ester acyltransferase mutant as described in claim 1; (4) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate described in form (3).
7. The use of an amino acid ester acyltransferase mutant as described in claim 1 or 2, or an amino acid ester acyltransferase mutant catalyst as described in claim 6, in catalyzing the condensation reaction of L-alanine methyl ester and L-glutamine to generate alanine-glutamyl dipeptide.
8. The application according to claim 7, characterized in that, The application involves adding the amino acid ester acyltransferase mutant catalyst as described in claim 1 or 2, or the amino acid ester acyltransferase mutant catalyst as described in claim 6, to a buffer solution containing L-alanine methyl ester and L-glutamine to catalyze the synthesis of alanine-glutamyl dipeptide from L-alanine methyl ester and L-glutamine.
9. The application according to claim 7 or 8, characterized in that, The reaction temperature is 20-40°C, and the reaction pH is 7.0-9.
0.
10. The application according to claim 7 or 8, characterized in that, The concentrations of the substrate L-alanine methyl ester were 100 mM to 1000 mM, and the concentrations of the substrate L-glutamine were 100 mM to 1000 mM.