A transferase and a method for producing carnosine by green enzyme

Abstract

This application provides a transferase and a green enzymatic method for producing carnosine. By performing single- or double-point mutations at position 86 (E86V) and position 259 (D259A) of the wild-type DmpA enzyme, highly active mutants DmpA-2 and DmpA-6 are obtained, significantly improving the catalytic efficiency of β-amino acid ester acyltransferases. This method uses β-alanine and L-histidine as substrates, utilizes recombinant bacteria to express the mutant enzyme, and catalyzes the synthesis of carnosine under mild conditions. The product concentration reaches a maximum of 5.36 g / L, which is 3.53 times that of the wild type. It has the advantages of green reaction conditions and high product purity, making it suitable for industrial production.

Description

Technical Field

[0001] This application belongs to the field of synthetic biology, specifically relating to a transferase and a green enzymatic method for producing carnosine. Background Technology

[0002] L-Carnosine (β-alanyl-L-histidine) is a naturally occurring dipeptide composed of β-alanine and L-histidine linked by peptide bonds. It possesses various biological activities, including antioxidant, anti-aging, anti-fatigue, and immunomodulatory effects, and has broad application prospects in medicine, food, and cosmetics. It is widely present in vital tissues such as muscles, brain, and heart, playing a crucial role in maintaining normal physiological functions. In the pharmaceutical field, L-carnosine can be used to treat various diseases, such as hypertension, heart disease, age-related cataracts, and ulcers, and also has adjuvant therapeutic effects in anti-tumor treatment and wound healing. In the food industry, it can be used as a natural antioxidant and food additive to extend shelf life and improve food quality. In the cosmetics industry, it can delay skin aging and reduce wrinkle formation.

[0003] Currently, the main methods for producing L-carnosine are chemical synthesis and biosynthesis. Chemical synthesis has many drawbacks, such as cumbersome steps, complex protection and deprotection of the active groups of the substrate, and harsh reaction conditions, often requiring high temperature, high pressure, strong acids or strong bases, resulting in low yield, high cost, significant pollution, and difficulty in guaranteeing product purity. Biosynthesis, on the other hand, has significant advantages. Reaction conditions are mild, typically carried out at room temperature and pressure in an aqueous phase, conforming to green chemistry principles, and producing products with high optical purity. Enzymatic synthesis is an important biosynthetic route, but existing technologies have some problems. For example, the L-carnosine synthesis reaction catalyzed by aminopeptidase requires activation of the carboxyl group of β-alanine or the amino group of L-histidine, which is complex and involves environmental pollution and increased costs. While the reverse hydrolysis reaction catalyzed by carnosine hydrolase does not require substrate activation, the reported β-amino acid ester acyltransferases are few in variety and have low activity, resulting in long reaction times and low product concentrations, making it difficult to meet industrial requirements. Summary of the Invention

[0004] To address the shortcomings of existing technologies, such as the limited variety of β-amino acid ester acyltransferases and low product concentrations, this application proposes a transferase and a green enzymatic method for producing carnosine. By modifying wild-type DmpA to obtain a mutant, the activity of β-amino acid ester acyltransferase is improved, thereby increasing the concentration of the product L-carnosine.

[0005] This application proposes a transferase, wherein the transferase is a mutant obtained by site-directed modification of wild-type DmpA; wherein the mutant is a DmpA-2 mutant and a DmpA-6 mutant.

[0006] Preferably, the wild-type DmpA is derived from *Escherichia coli*, which is preserved at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beifu West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a preservation date of April 28, 2024, and is classified and named *Escherichia coli*. Escherichia coli The registration number is: CGMCC No.30472.

[0007] Preferably, the wild-type DmpA amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.9:

[0008] SEQ ID NO.1:

[0009] MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGSGELTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLK GGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKDGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV.

[0010] SEQ ID NO.9:

[0011]

[0012] Preferably, the DmpA-2 mutant is formed by mutating glutamic acid at position 86 of the DmpA sequence to valine.

[0013] Preferably, the DmpA-6 mutant is formed by mutating glutamic acid at position 86 of the DmpA sequence to valine and aspartic acid at position 259 to alanine.

[0014] Preferably, the amino acid sequence of the DmpA-2 mutant is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.10.

[0015] Preferably, the amino acid sequence of the DmpA-6 mutant is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.11.

[0016] This application also discloses a green enzymatic method for producing carnosine, characterized in that the production method includes:

[0017] (1) Using β-alanine and L-histidine as substrates, the wild-type DmpA was molecularly docked with AutoDockVina, and mutation sites were screened to obtain mutants.

[0018] (2) The mutant gene obtained by screening was cloned into the expression vector, transformed into Escherichia coli BL21, and expressed under IPTG induction to obtain recombinant bacteria;

[0019] (3) The above recombinant bacteria were fermented to obtain an active β-amino acid ester acyltransferase cell suspension;

[0020] (4) Centrifuge the above β-amino acid ester acyltransferase cell suspension at 10,000 rpm for 1 min to remove culture medium residue, carefully discard the supernatant, add 1 mL PBS and gently pipette, centrifuge again at 10,000 rpm for 5 min, take the supernatant to obtain β-amino acid ester acyltransferase solution, catalyze β-alanine and L-histidine in the β-amino acid ester acyltransferase solution under specific conditions to obtain green enzymatic carnosine, and quantitatively detect the concentration of green enzymatic carnosine.

[0021] Preferably, in step (4), the specific conditions are a temperature of 35-37°C, a rotation speed of 220 rpm, and a reaction time of 20 min.

[0022] Preferably, in step (4), the concentration detection is performed by high performance liquid chromatography.

[0023] The beneficial effects of the embodiments of this application are as follows: the mutant obtained by site-directed mutagenesis of wild-type DmpA enzyme uses substrates derived from green fermentation, which significantly improves the catalytic activity of β-amino acid ester acyltransferase, thereby enhancing carnosine synthesis efficiency and significantly increasing product concentration, and has broad prospects for industrial development and application. Attached Figure Description

[0024] Figure 1 This is a diagram of the AutoDockVina docking configuration in Embodiment 1 of this application;

[0025] Figure 2 This is a schematic diagram of the enzymatic reaction in Example 4 of this application;

[0026] Figure 3 This is the molecular formula diagram of carnosine produced by the green enzymatic method in Example 4 of this application. Detailed Implementation

[0027] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0028] Example 1:

[0029] Screening for mutation points;

[0030] Gene and protein characteristics: Wild-type DmpA belongs to the β-aminopeptidase family and is derived from Escherichia coli. Escherichia coli It is preserved at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beifu West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a preservation date of April 28, 2024, and is classified and named as: *Escherichia coli*. Escherichia coli The enzyme is registered under the CGMCC No. 30472. It can specifically hydrolyze β-alanyl-L-histidine. The crystal structure of this enzyme is a four-layer αββα architecture. The core active site is formed around residues such as Ser250, Glu144, and Asp290. These structural studies show that the active pocket is composed of several key side chains, which determine the substrate binding mode.

[0031] AutoDockVina docking results: docking was performed using β-alanyl-L-histidine as a substrate, such as... Figure 1The optimal conformation shown indicates that its carboxyl and hydroxyl groups form stable hydrogen bonds with S84, E86, and D259 on the surface of the DmpA protein, respectively. These three residues are located in the entrance region of the active pocket, adjacent to known catalytic residues, and can provide a hydrogen bond network and charge compensation, thereby enhancing substrate localization. Specifically, S84 provides a polar side chain hydroxyl group, participating as a hydrogen bond acceptor for the substrate carboxyl group; E86, with a negatively charged side chain, can form a salt bridge with the positively charged amino group of L-carnosine, further immobilizing the substrate; and D259, located deep within the active pocket, forms additional hydrogen bonds to maintain the substrate posture. Therefore, site-directed mutagenesis of S84, E86, and D259 is recommended.

[0032] The DmpA-2 mutant is formed by mutating glutamic acid at position 86 of the DmpA sequence to valine. Its amino acid sequence is shown in SEQ ID NO.2, and its corresponding nucleotide sequence is shown in SEQ ID NO.10.

[0033] The DmpA-6 mutant is formed by mutating glutamic acid at position 86 of the DmpA sequence to valine and aspartic acid at position 259 to alanine. Its amino acid sequence is shown in SEQ ID NO.3 and its corresponding nucleotide sequence is shown in SEQ ID NO.11.

[0034] The DmpA-3 mutant is formed by mutating the aspartic acid at position 259 of the DmpA sequence to alanine. Its amino acid sequence is shown in SEQ ID NO.4, and its corresponding nucleotide sequence is shown in SEQ ID NO.12.

[0035] The DmpA-4 mutant is formed by mutating serine at position 84 of the DmpA sequence to phenylalanine and glutamic acid at position 86 to valine. Its amino acid sequence is shown in SEQ ID NO.5 and its corresponding nucleotide sequence is shown in SEQ ID NO.13.

[0036] The DmpA-5 mutant is formed by mutating serine at position 84 of the DmpA sequence to phenylalanine and aspartic acid at position 259. Its amino acid sequence is shown in SEQ ID NO.6 and its corresponding nucleotide sequence is shown in SEQ ID NO.14.

[0037] The DmpA-1 mutant is formed by mutating serine at position 84 of the DmpA sequence to phenylalanine. Its amino acid sequence is shown in SEQ ID NO.7, and its corresponding nucleotide sequence is shown in SEQ ID NO.15.

[0038] The DmpA-7 mutant is formed by mutating serine at position 84 of the DmpA sequence to phenylalanine, glutamic acid at position 86 to valine, and aspartic acid at position 259 to alanine. Its amino acid sequence is shown in SEQ ID NO.8, and the corresponding nucleotide sequence is shown in SEQ ID NO.16.

[0039] SEQ ID NO.2:

[0040] MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGSGVLTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLK GGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKDGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0041] SEQ ID NO.3: MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGSGVLTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHG LKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKAGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0042] SEQ ID NO.4:MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGSGELTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKAGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0043] SEQ ID NO.5:MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGFGVLTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKDGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0044] SEQ ID NO.6:MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGFGELTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKAGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0045] SEQ ID NO.7:MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGFGELTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKDGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0046] SEQ ID NO.8:MRVQLSPEQVPRRMRIRELLPDLDLGAYPPGPLNSITDVPGVHVHTQEIFGAQGAINTGVTCIVPRPNWSTNACYAGVFRFNGFGVLTGAHLIEETGLLCSPIVLTGTFNIGAAHQGIYQYAVKHLGTNKDGQLEWLMLPVVGETFDGYLHDCTSFAVAPAHIVHGLESVVAGEPVREGNVGGGVGMVCHGLKGGTGSSSRQVLGTYTVAALVQANYGQLRDLRIAGVPVGKILTEDAASDPSRQGMYEEVAQAKAEKAGSIIVVLATDAPLHPAQLQRVAKRATVGLARVGGQGHNLSGDIFLAFSTGNEIPVNQHKRPASVARTIDVLDDSALNTLFEATADAVEEAIYNALCMAESLQGFQGHTIEALPLARLKEIMRQYQRV;

[0047] SEQ ID NO.10:

[0048]

[0049] SEQ ID NO.11:

[0050]

[0051] SEQ ID NO.12:

[0052]

[0053] SEQ ID NO.13:

[0054]

[0055] SEQ ID NO.14:

[0056]

[0057] SEQ ID NO.15:

[0058]

[0059] SEQ ID NO.16:

[0060]

[0061] Example 2:

[0062] Construction of recombinant bacteria;

[0063] (1) Preparation of LB medium (1L):

[0064] 1% tryptone, 0.5% yeast extract, 1% sodium chloride, deionized water to make up the difference, autoclave at 126℃ for 20 minutes.

[0065] (2) Preparation of Escherichia coli BL21 competent cells:

[0066] Freshly activated single colonies of E. coli BL21 were picked from LB agar plates and inoculated into 5 ml of LB liquid medium. The culture was incubated at 37°C with shaking for approximately 12 h until the late logarithmic growth phase. The bacterial suspension was then inoculated into 100 ml of LB liquid medium at a ratio of 1:100 and incubated at 37°C with shaking for 2–3 h until the OD600 reached approximately 0.5. The culture was then transferred to centrifuge tubes and placed on ice for 10 min. The tubes were then centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was discarded. The cells were gently resuspended in 10 ml of pre-chilled 0.05 mol / L CaCl2 solution and placed on ice for 15–30 min. The cells were then centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was discarded. 4 ml of pre-chilled 0.05 mol / L CaCl2 solution containing 15% glycerol was added, and the cells were gently resuspended. The cells were then placed on ice for 5 min to obtain a competent cell suspension. The competent cells were aliquoted into 200 μL portions and stored at -80°C.

[0067] (3) Plasmid transformation:

[0068] Remove competent cells from the -80℃ freezer and thaw them on ice. Add the plasmid DNA to be transformed and incubate on ice for 30 min. At the same time, turn on the water bath and adjust it to 42℃. After the ice bath, remove the competent cells containing DNA from the ice and heat shock them at 42℃ for 90 s. Then quickly incubate on ice for about 1 min, add about 800 μL of LB (preheated at 37℃ for 1 min), and revive at 37℃ and 220-250 rpm for 40 min. After revival, centrifuge at 4000 rpm for 5 min to collect the cells, then resuspend them in 100 μL of antibiotic-free LB and spread them on the appropriate selective LB plates for overnight culture.

[0069] The wild-type CAR001 strain was obtained by transforming plasmid pET28a-DmpA into BL21; the recombinant strain CAR002 was obtained by transforming plasmid pET28a-DmpA-S84F into BL21; the recombinant strain CAR003 was obtained by transforming plasmid pET28a-DmpA-E86V into BL21; the recombinant strain CAR004 was obtained by transforming plasmid pET28a-DmpA into BL21; and the recombinant strain CAR004 was obtained by transforming plasmid pET28a-DmpA into BL21. Transforming the plasmid pET28a-DmpA-S84F-D259A into BL21 yields the recombinant strain CAR005; transforming the plasmid pET28a-DmpA-S84F-D259A into BL21 yields the recombinant strain CAR006; transforming the plasmid pET28a-DmpA-E86V-D259A into BL21 yields the recombinant strain CAR007; transforming the plasmid pET28a-DmpA-S84F-E86V-D259A into BL21 yields the recombinant strain CAR008.

[0070] The characteristics of the strains and plasmids used are shown in Table 1.

[0071] .

[0072] Example 3:

[0073] Preparation of β-amino acid ester acyltransferase cell suspension:

[0074] (1) Prepare LB medium containing kanamycin: Take 100 mL of LB medium from Example 2 and add kanamycin to make the final concentration of kanamycin 50 µg / mL;

[0075] (2) Preparation of seed culture: Single positive clones from each of the recombinant strains CAR001, CAR002, CAR003, CAR004, CAR005, CAR006, CAR007, and CAR008 were picked and inoculated into 5 mL of LB medium containing 50 µg / mL kanamycin; the medium was incubated at 37°C with shaking at 220–250 rpm for 12 h until the medium became turbid. Once the culture medium reaches the late logarithmic growth phase of 0.8–1.0, it becomes the seed culture.

[0076] (3) Pre-culture: Take a 1L Erlenmeyer flask, add 100mL of pre-prepared LB medium containing 50µg / mL kanamycin, and inoculate the seed culture at a volume ratio of 5% (i.e., 5mL). Mix well and continue to incubate at 37°C and 220–250rpm with shaking for 2–3 hours until the medium reaches the desired concentration. When the value reaches 0.5, the cell is in the early stage of the logarithmic growth phase.

[0077] (4) IPTG induction: Prepare 1M IPTG stock solution in advance (dissolve and filter with sterile water), and preheat it in a 37°C water bath for 1 min before use; add 7.3µL of 1M IPTG to 100mL of culture medium to make the final IPTG concentration 0.2mmol / L; after adding IPTG, immediately lower the culture temperature to 30°C and continue to culture at 220-250rpm for 16h to obtain the corresponding β-amino acid ester acyltransferase cell suspension; low temperature induction helps the target protein maintain solubility, and prolonging the expression time can significantly increase the yield.

[0078] Example 4:

[0079] Catalytic synthesis of green enzymatic carnosine:

[0080] (1) Solution preparation:

[0081] To prepare 50mM Tris-HCl (pH 7.5): Weigh 6.06g Tris (molecular weight 121.14g / mol) and dissolve it in about 800mL of deionized water. Adjust the pH to 7.5 dropwise with 1M HCl, and then bring the volume to 1L with deionized water.

[0082] Preparation of substrate solution (pH 9.0): To prepare a substrate solution containing 120 mM β-alanine methyl ester hydrochloride + 80 mM L-histidine: Weigh 0.8375 g of β-alanine methyl ester hydrochloride and 0.62 g of L-histidine, place them in a 50 mL centrifuge tube, add 20 mL of deionized water to dissolve them, adjust the pH to 9.0 using 10% NaOH, and bring the volume to 50 mL with deionized water.

[0083] β-Amino acid ester acyltransferase solution: Take 1 mL of each of the β-amino acid ester acyltransferase cell suspensions prepared in Example 2, centrifuge at 10000 rpm for 1 min to remove culture medium residue, carefully discard the supernatant, add 1 mL of PBS (pH 7.4), gently pipette to obtain enzyme lysis buffer, centrifuge again at 10000 rpm for 5 min, and take the supernatant as the β-amino acid ester acyltransferase solution.

[0084] (2) Enzymatic reaction: Take 1 mL of substrate solution in a 2 mL centrifuge tube, add 0.1 mL of β-amino acid ester acyltransferase solution, and react for 20 min at 220 rpm on a 37℃ constant temperature shaker; at the same time, a blank control is set up: 1 mL of substrate + 0.1 mL of PBS is treated under the same conditions. The schematic diagram of the enzyme-catalyzed reaction is shown in the figure. Figure 2 As shown.

[0085] (3) Reaction termination: Place the reaction tube (containing the sample and blank) directly into a boiling water bath (100℃) for 5 minutes to stop the enzyme reaction. Then, cool it to room temperature in an ice bath to prevent thermal degradation, yielding green enzymatic carnosine. The carnosine molecular formula is as follows: Figure 3 As shown.

[0086] Example 5:

[0087] Green enzymatic method for detecting carnosine concentration and enzyme activity:

[0088] (1) High-performance liquid chromatography (HPLC) was used to detect carnosine concentration using a green enzyme method:

[0089] Chromatographic conditions:

[0090] Chromatograph: Waters 2695;

[0091] Chromatographic column: C18 reverse phase column, 250 mm × 4.6 mm, 5 µm; column temperature: 25°C;

[0092] Mobile phase: A: 0.1M phosphate buffer (pH 3.45), B: acetonitrile ratio A:B = 80:20 (v / v);

[0093] Other parameters: flow rate 1.0 mL / min, detection wavelength 210 nm (UV), injection volume 20 µL; separation time 8 min.

[0094] Sample pretreatment:

[0095] Take 1 mL of green enzyme-processed carnosine and place it directly into a centrifuge tube;

[0096] Protein precipitation: Add an equal volume of 10% glacial ethyl acetate, incubate on ice for 5 min, centrifuge at 10000 rpm for 5 min, and collect the supernatant;

[0097] Filtration: Use a 0.22µm PTFE filter membrane to filter into the HPLC vial to prevent column clogging.

[0098] Experimental results:

[0099] The carnosine concentrations were calculated using high-performance liquid chromatography (HPLC): 1.52 g / L for wild-type strain CAR001; 1.3 g / L for CAR002; 2.68 g / L for CAR003; 1.3 g / L for CAR004; 0.4 g / L for CAR005; 0.21 g / L for CAR006; and 5.36 g / L for CAR007. Under the same culture conditions, CAR007 produced 3.53 times more carnosine than wild-type strain CAR001, and CAR003 produced 1.76 times more. The carnosine purity of all strains CAR001-CAR007 was >99%. The relative enzyme activity of CAR007 was 3.53 times that of wild-type CAR001, and the relative enzyme activity of CAR003 was 1.76 times that of wild-type CAR001.

[0100] In summary, by designing and modifying DmpA transferase, mutants DmpA-2 (E86V) and DmpA-6 (E86V / D259A) with significantly enhanced catalytic performance were successfully obtained, and a highly efficient and green enzymatic process for the synthesis of carnosine was established. This process, conducted under mild conditions, uses β-alanine and L-histidine as substrates and utilizes a highly active mutant enzyme expressed by engineered bacteria for catalysis, achieving a maximum carnosine yield of 5.36 g / L, 3.53 times that of the original wild type. It effectively overcomes the shortcomings of traditional chemical synthesis and existing enzymatic processes, offering significant advantages such as simple steps, mild conditions, high product concentration, and environmental friendliness, providing a reliable and promising technical solution for the industrial production of carnosine.

[0101] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A transferase, characterized in that, The transferase is a mutant obtained by site-directed modification of wild-type DmpA; wherein, the mutant is DmpA-2 mutant and DmpA-6 mutant; the amino acid sequence of wild-type DmpA is shown in SEQ ID NO.1; the DmpA-2 mutant is obtained by mutating glutamic acid at position 86 of the DmpA sequence to valine; the DmpA-6 mutant is obtained by mutating glutamic acid at position 86 of the DmpA sequence to valine and aspartic acid at position 259 to alanine; the amino acid sequence of the DmpA-2 mutant is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.10; the amino acid sequence of the DmpA-6 mutant is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.11.

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