Application of alpha-glutamate ligase GHU in soybean protein-mediated catalytic synthesis of proline-threonine dipeptide

Abstract

The application of alpha-glutamate ligase GHU in soybean protein-mediated catalytic synthesis of proline-threonine dipeptide belongs to the technical field of biotechnology. In order to solve the technical problems that there is no specific and efficient biological catalytic method for proline-threonine dipeptide in the prior art, and it is difficult to realize the directional synthesis of the dipeptide, the coding gene of alpha-glutamate ligase GHU is constructed into a recombinant expression vector, which is transformed into E. coli to induce expression to obtain enzyme protein. Proline and threonine are used as substrates to generate proline-threonine dipeptide through enzymatic reaction. The experimental results show that the enzyme has significant catalytic preference for Pro-Thr, the reaction condition is mild and environmentally friendly, and the cumbersome steps and pollution problems of traditional chemical synthesis are avoided. The application provides a new enzyme resource and technical path for the green enzyme synthesis of functional dipeptide, and has important application value in the fields of food, medicine, health care products and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of α-glutamate ligase GHU in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides. Background Technology

[0002] Dipeptides are small peptide compounds formed by two amino acids linked by peptide bonds. Although dipeptides have relatively simple structures, different amino acid compositions and sequences can endow them with different physicochemical properties and biological functions, thus possessing broad development value in the fields of medicine, food, nutritional supplements, and cosmetics. For example, aspartame (L-aspartic-L-phenylalanine methyl ester) is a typical example of the development and application of dipeptide derivatives. Modified from its dipeptide backbone, it exhibits a significant sweetness and has been widely used as a food sweetener. This demonstrates that dipeptides can not only serve as nutritional or functional components but also exhibit distinct sensory characteristics due to their specific structures. Their small molecular weight and relatively simple structure often make them more likely to exhibit specific flavor characteristics in food systems. Proline-threonine dipeptide (Pro-Thr) is a type of umami dipeptide with the potential to be screened and developed as a novel umami molecule or functional peptide. Research on its flavor characteristics and related functional activities is expected to provide new candidate molecules for food flavor improvement and the development of functional small peptides.

[0003] Currently, dipeptide preparation mainly involves chemical synthesis and biocatalysis. While chemical synthesis has been used for a longer period and is relatively mature, it typically requires protection and deprotection of active groups such as amino and carboxyl groups. This process involves numerous steps, is relatively cumbersome, and may be accompanied by increased side reactions, complex separation and purification processes, and higher production costs. Especially when developing food, pharmaceutical, and functional products, traditional chemical routes still have limitations in terms of greenness, mildness, and high selectivity.

[0004] In contrast, enzymatic catalysis can directly utilize amino acid substrates under milder conditions, offering advantages such as high catalytic selectivity, relatively green reaction systems, and milder process conditions. Therefore, it has gradually become an important research direction for the preparation of targeted dipeptides. Among them, L-amino acid ligases (Lals) can directly catalyze peptide bond formation using free L-amino acids as substrates with the participation of ATP, avoiding complex substrate preactivation and multi-step protection operations, showing promising application prospects in dipeptide biosynthesis. However, existing Lals still have certain limitations in practical applications, such as limited recognition ability for specific substrate combinations, low target product generation efficiency, and decreased catalytic performance at higher substrate concentrations, thus restricting their further application in the targeted synthesis of specific functional dipeptides.

[0005] Among reported ATP-dependent amino acid ligases, RimK family enzymes have attracted attention due to their ability to catalyze amino acid ligation reactions. α-Glutamate ligases belong to this enzyme family and are widely found in bacteria and other microorganisms. Although RimK family proteins exhibit some sequence conservation, different members often show significant differences in substrate recognition preferences, active site microenvironment, and product formation characteristics. Therefore, high sequence homology does not necessarily indicate identical catalytic functions. Due to differences in substrate recognition characteristics and catalytic behavior among different family members, current technology lacks a systematic understanding of the types of substrates that α-glutamate ligases can catalyze and their application range in the synthesis of different dipeptides. Currently, there are no reports of their use in catalyzing the synthesis of proline-threonine dipeptides (Pro-Thr).

[0006] Therefore, developing an enzyme-catalyzed method that can efficiently and specifically catalyze the synthesis of Pro-Thr dipeptides under mild and environmentally friendly reaction conditions is of significant research importance and application potential. However, current technologies lack specific and efficient biocatalytic methods for Pro-Thr dipeptides. How to utilize ligases with specific substrate preferences to achieve the directed synthesis of Pro-Thr has become a pressing technical problem to be solved in this field. Summary of the Invention

[0007] To address the lack of specific and efficient biocatalytic methods for the proline-threonine dipeptide (Pro-Thr) in existing technologies, which hinders the targeted synthesis of this dipeptide, this invention constructs a recombinant expression vector encoding the α-glutamate ligase GHU, transforms it into *E. coli* to induce expression, and obtains the enzyme protein. Using proline and threonine as substrates, an enzymatic reaction is performed to generate the proline-threonine dipeptide. Experimental results show that this enzyme exhibits a significant catalytic preference for Pro-Thr, with mild reaction conditions and an environmentally friendly approach, avoiding the cumbersome steps and pollution problems of traditional chemical synthesis.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide the application of α-glutamate ligase GHU in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A second objective of this invention is to provide the application of the gene encoding the aforementioned α-glutamate ligase GHU in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

[0010] In one embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] A third objective of this invention is to provide the application of a recombinant expression vector containing the above-mentioned genes in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

[0012] In one embodiment of the present invention, the vector backbone of the recombinant expression vector is pET-21a(+).

[0013] A fourth objective of this invention is to provide the application of recombinant bacteria containing the above-mentioned genes or the above-mentioned recombinant expression vectors in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

[0014] In one embodiment of the present invention, the host cell of the recombinant bacteria is *Escherichia coli*. C43(DE3) .

[0015] The fifth objective of this invention is to provide a method for catalytic synthesis of proline-threonine dipeptides, wherein the method uses proline and threonine as substrates and performs an enzymatic reaction in the presence of α-glutamate ligase GHU to generate proline-threonine dipeptides; the amino acid sequence of the α-glutamate ligase GHU is shown in SEQ ID NO.1.

[0016] In one embodiment of the present invention, the enzymatic reaction conditions are pH 7.4, temperature 35°C, and reaction time 12-16 h.

[0017] In one embodiment of the present invention, the α-glutamate ligase GHU is obtained by inoculating recombinant Escherichia coli overexpressing the α-glutamate ligase GHU gene into 2×TY-Soya medium and adding IPTG to induce expression; the nucleotide sequence of the α-glutamate ligase GHU gene is shown in SEQ ID NO.2; the 2×TY-Soya medium consists of 16 g / L soybean protein hydrolysate, 10 g / L yeast extract, 5 g / L sodium chloride, and the remainder is water.

[0018] The beneficial effects of this invention are: This invention marks the first successful targeted catalytic synthesis of the proline-threonine dipeptide (Pro-Thr) by the α-glutamate ligase GHU. Experiments have demonstrated that this enzyme exhibits a significant catalytic preference and specific synthetic characteristics for Pro-Thr, filling a technological gap in this field.

[0019] This invention provides a novel, environmentally friendly method for the synthesis of dipeptides with mild reaction conditions. Compared with traditional solid-phase chemical synthesis methods, this invention eliminates the need for protection and deprotection of amino and carboxyl groups, produces no organic waste liquid or racemic products, and operates under mild reaction conditions (pH 7.4, 35°C), meeting the requirements of green chemistry and biomanufacturing.

[0020] The α-glutamate ligase GHU used in this invention can efficiently synthesize target dipeptides in an in vitro reaction system using free proline and threonine as substrates. Comparative experiments show that the enzyme's catalytic activity for Pro-Thr is significantly superior to its activity for other substrate combinations, demonstrating unique substrate recognition specificity.

[0021] This invention is not only applicable to the preparation of Pro-Thr dipeptide, but also provides a theoretical basis for substrate-specific research and targeted modification of other RimK family ligases, and has broad application prospects. Attached Figure Description

[0022] Figure 1 The image shows the identification results of E. coli transformed with the pET-21a(+)-GHU plasmid; among them, Figure 1 A in the text represents recombinant Escherichia coli. C43(DE3)-GHU Figure showing growth on 2×TY-Soya solid medium containing ampicillin resistance. Figure 1 B in the text represents the blank recipient *E. coli*. C43(DE3) Figure showing growth on 2×TY-Soya solid medium containing ampicillin resistance; Figure 2 The figure shows the effect of different culture medium compositions on GHU protein expression levels; where 1-7 are LB medium, LB-Soya medium, 2×TY medium, 2×TY-Soy medium, 2×TY-Casein medium, 2×TY-Gelatin medium and 2×TY-Pea medium, respectively. Figure 3 This is a colorimetric graph showing the results of determining the concentration of purified protein using the BCA method; where AH and 1-12 represent positions for easy description in the experimental results. Figure 4 The image shows the SDS-PAGE electrophoresis results of the purified GHU protein; among them, Figure 4 In the diagram, A represents the control group, and lanes 1-10 correspond to the protein molecular weight standard, soluble protein solution, insoluble precipitate, nickel column flow-through buffer, and gradient wash buffers 1-6, respectively. Figure 4 In the diagram, B represents the SDS-PAGE electrophoresis result of the target protein sample eluted by gradient elution. Lane 1 represents the protein molecular weight standard, and lanes 2-10 represent the eluted components obtained under different column volumes. Figure 5 The extracted ion chromatogram of proline-threonine in the supernatant of the reaction solution is shown. Figure 6 This is a low-energy and high-energy channel mass spectrum of proline-threonine in the supernatant of the reaction solution. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0025] Escherichia coli DH5α was purchased from Beijing Baosai Biotechnology Co., Ltd.; Escherichia coli C43(DE3) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0026] The composition of LB medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water.

[0027] The composition of LB-Soya medium is: 10 g / L soybean protein hydrolysate, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water.

[0028] The composition of 2×TY medium is: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and the balance is water.

[0029] The composition of 2×TY-Soya medium: 16 g / L soybean protein hydrolysate, 10 g / L yeast extract, 5 g / L sodium chloride, and the balance being water.

[0030] The composition of 2×TY-Casein medium is: 16 g / L casein peptone, 10 g / L yeast extract, 5 g / L sodium chloride, and the balance is water.

[0031] The composition of 2×TY-Gelatin medium: 16 g / L gelatin peptone, 10 g / L yeast extract, 5 g / L sodium chloride, and the balance being water.

[0032] The composition of 2×TY-Pea medium: 16 g / L pea protein hydrolysate, 10 g / L yeast extract, 5 g / L sodium chloride, and the balance being water.

[0033] All culture media used in this invention require the addition of 50 μg / mL ampicillin after sterilization to maintain the plasmid.

[0034] The culture medium and buffer solution used in this invention must be adjusted to pH 7.4 and filtered through a 0.22 μm filter membrane (select either aqueous or organic based on the solvent type).

[0035] The amino acid sequence of the α-glutamate ligase GHU in this invention is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1: MKIAILSNGPANYATKRIKEVAIARGHDAEVVRYDDCYAIVKQDNPVVMKSGEPLSGYDAVMPFFAPRMTRYASAIIRQFENQGIFTMTRSIAVVRANDKLRSTQIMAKHGIDMPVTAVSRNTADIDSLIDDVGGTPVIIKLARGTQGNGVV LAETKKAARSALQALYIYNDDDGTNLLVQEYIEESAGTDIRVFVVGGRVVASMKRQSLNDDFRSNLHKGGEGTIIKLTEEERKIAIKATKALGLNFAGVDLMRSKRGPLVLEVNASPGFGIEDVTGRDVATPILEYIERNAMKGVKKDKIGA; SEQ ID NO.2: ATGAAAATCGCTATTCTGTCTAACGGTCCGGCGAACTATGCAACCAAACGTATTAAAGAAGTTGCAATCGCGCGTGGTCACGATGCAGAAGTTGTTCGTTATGATGATTGCTACGCAATCGTTAAACAGGATAACCCGGTTGTGATGAAATCTGGCGAACCGCTGTCCGGTTACGATGCGGTTATGCCGTTCTTCGCGCCGCGTATGACCCGTTACGCGTCCGCTATCATCCGTCAGTTCGAAAACCAGGGTATCTTCACCATGACCCGTAGCATCGCGGTTGTTCGTGCTAACGATAAACTGCGTTCTACCCAGATCATGGCGAAACACGGTATCGATATGCCGGTTACCGCGGTTAGCCGTAACACCGCGGATATTGATTCTCTGATCGATGATGTTGGTGGTACCCCGGTTATCATCAAACTGGCGCGTGGCACCCAGGGTAACGGCGTTGTGCTGGCGGAAACCAAAAAAGCTGCGCGTAGCGCGCTGCAGGCACTGTACATCTACAACGATGATGGTACTAACCTGCTGGTTCAGGAATACATCGAAGAATCTGCGGGTACCGATATCCGTGTTTTCGTTGTTGGTGGTCGTGTTGTTGCTAGCATGAAACGTCAGTCTCTGAACGATGATTTCCGTTCTAACCTGCACAAAGGTGGTGAAGGTACCATTATCAAACTGACCGAAGAAGAACGTAAAATCGCGATCAAAGCGACCAAAGCTCTGGGTCTGAACTTCGCGGGCGTTGATCTGATGCGTAGCAAACGTGGTCCGCTGGTTCTGGAAGTTAACGCAAGCCCAGGTTTCGGTATCGAAGATGTTACCGGCCGTGATGTTGCGACCCCGATCCTGGAATACATTGAACGTAACGCGATGAAAGGTGTTAAAAAAGATAAAATCGGTGCG。

[0037] Example 1: Preparation of α-glutamate ligase GHU 1. Construction of recombinant vector and recombinant bacterium containing α-glutamate ligase GHU gene Based on the amino acid sequence of the α-glutamate ligase GHU, codon optimization was performed on E. coli to obtain the coding sequence (excluding the start codon, as shown in SEQ ID NO.2). To facilitate protein purification and subsequent cloning, a 6×His tag coding sequence and an NcoI restriction site were sequentially introduced at the 5' end of this coding sequence, and a HindIII restriction site was introduced at the 3' end, thereby obtaining the complete artificially synthesized coding sequence (as shown in SEQ ID NO.3).

[0038] SEQ ID NO.3: CCATGG GCAGCAGCCATCACCATCATCACCACATGAAAATCGCTATTCTGTCTAACGGTCCGGCGAACTATGCAACCAAACGTATTAAAGAAGTTGCAATCGCGCGTGGTCACGATGCAGAAGTTGTTCGTTATGATGATTGCTACGCAATCGTTAAACAGGATAACCCGGTTGTGATGAAATCTGGCGAACCGCTGTCCGGTTACGATGCGGTTATGCCGTTCTTCGCGCCGCGTATGACCCGTTACGCGTCCGCTATCATCCGTCAGTTCGAAAACCAGGGTATCTTCACCATGACCCGTAGCATCGCGGTTGTTCGTGCTAACGATAAACTGCGTTCTACCCAGATCATGGCGAAACACGGTATCGATATGCCGGTTACCGCGGTTAGCCGTAACACCGCGGATATTGATTCTCTGATCGATGATGTTGGTGGTACCCCGGTTATCATCAAACTGGCGCGTGGCACCCAGGGTAACGGCGTTGTGCTGGCGGAAACCAAAAAAGCTGCGCGTAGCGCGCTGCAGGCACTGTACATCTACAACGATGATGGTACTAACCTGCTGGTTCAGGAATACATCGAAGAATCTGCGGGTACCGATATCCGTGTTTTCGTTGTTGGTGGTCGTGTTGTTGCTAGCATGAAACGTCAGTCTCTGAACGATGATTTCCGTTCTAACCTGCACAAAGGTGGTGAAGGTACCATTATCAAACTGACCGAAGAAGAACGTAAAATCGCGATCAAAGCGACCAAAGCTCTGGGTCTGAACTTCGCGGGCGTTGATCTGATGCGTAGCAAACGTGGTCCGCTGGTTCTGGAAGTTAACGCAAGCCCAGGTTTCGGTATCGAAGATGTTACCGGCCGTGATGTTGCGACCCCGATCCTGGAATACATTGAACGTAACGCGATGAAAGGTGTTAAAAAAGATAAAATCGGTGCGAAGCTT (The underlined part is the protective base, and the bold part is the 6×His tag coding sequence).

[0039] The obtained complete synthetic coding sequence was double-digested with NcoI and HindIII, and the digestion products were recovered to obtain the GHU gene fragment. Simultaneously, the pET-21a(+) empty vector was double-digested with NcoI and HindIII, and the digestion products were recovered to obtain the linearized vector fragment. Subsequently, the digested GHU gene fragment was ligated to the linearized vector fragment using T4 DNA ligase to construct the recombinant plasmid pET-21a(+)-GHU. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL ampicillin, and incubated overnight at 37°C with inverted incubation. The next day, single colonies were picked for colony PCR identification to screen for recombinant clones. Positive clones were inoculated into liquid LB agar containing 50 μg / mL ampicillin and incubated overnight at 37°C with shaking. The recombinant plasmid was extracted using a plasmid extraction kit and sequenced by Sanger sequencing. Select the clone with the correct sequencing results, extract the recombinant plasmid using a plasmid extraction kit, and obtain the successfully constructed pET-21a(+)-GHU.

[0040] The successfully constructed pET-21a(+)-GHU plasmid was transformed into the E. coli expression host using the heat shock method. C43 (DE3) In the process, recombinant expression strains were obtained. C43(DE3)-GHU It is stored for a long time at -80°C containing glycerol.

[0041] Depend on Figure 1 It can be seen that recombinant Escherichia coli C43(DE3)-GHU It grows normally on resistant plates, while wild-type Escherichia coli... C43(DE3) They cannot grow due to the presence of antibiotics.

[0042] 2. Induction, expression, and purification of α-glutamate ligase GHU The specific method for inducing expression is as follows: Glycerol-preserved strains C43(DE3)-GHU Activated to the exponential phase (OD) in LB medium. 600 =0.4-0.6), inoculated into LB medium at a volume ratio of 1:40, cultured at 37℃ and 220 rpm for 3.5 h with shaking, cooled to 16℃ and then 0.5 mM IPTG was added, and expression was induced at 16℃ for 16 h.

[0043] Rapid purification was achieved using a Ni-NTA affinity chromatography column with the His tag added during plasmid construction. The specific steps are as follows: The induced bacterial culture was collected, centrifuged at 5000 rpm for 10 min at 4°C, and the precipitate was collected. Each gram of precipitate was resuspended in 10 mL of binding buffer, followed by the addition of 0.2 mg / mL lysozyme, 20 μg / mL DNase, 1 mM MgCl2, and 1 mM MPMSF. The mixture was shaken at 4°C for 30 min to digest. Subsequently, the lysis buffer was sonicated for 20 min, the pH of the lysis buffer was adjusted to 7.4, and the mixture was centrifuged at 12000 rpm for 25 min. The supernatant was collected, and a 1:1 ratio of binding buffer was added. The mixture was then filtered through a 0.22 µm aqueous filter to prepare the protein loading buffer. Prepare a pre-packed Ni-NTA column of the required loading capacity (5 mL of packing material can generally purify 100 mL of protein expressed in bacterial culture). Equilibrate the column with twice the column volume of Binding Buffer. Then, gradually add the protein loading solution to the column, collecting the flow-through and repeating the loading to increase protein binding. Wash the column with twice the column volume of Wash Buffer and collect the eluent. Repeat the washing step until the eluent reaches the baseline absorbance at 280 nm. Elute the His-Tag protein on the column with twice the column volume of Elution Buffer. Repeat this step twice, saving the eluent separately until the eluent reaches the baseline absorbance at 280 nm. Determine the concentration of the eluted protein using the BCA method, measuring its absorbance at 570 nm. All purification and centrifugation operations in the above process must be performed at 4°C.

[0044] 3. Optimization of the induction and expression of α-glutamate ligase GHU To optimize the strain C43(DE3)-GHU The growth status thus improves GHU The expression level of recombinant proteins was determined by adjusting the formulation of LB medium and 2×TY medium (as shown in Table 1). Glycerol-preserved strains... C43(DE3)-GHU Activated to the exponential phase (OD) in LB medium. 600 =0.4-0.6), inoculated into each modified medium at a volume ratio of 1:40, and cultured at 37℃ and 220 rpm for 3.5 h with shaking. After cooling to 16℃, 0.5 mM IPTG was added for low-temperature induction for 16 h. The control group used the original LB medium. The protein expression level was compared with the amount of purified GHU protein obtained after induction per 100 mL of medium. The purification method was the same as in step 2. The optimization results are shown in […]. Figure 2 .

[0045] Table 1 Culture medium improvement scheme

[0046] like Figure 2As shown, different culture medium compositions significantly affected the expression level of recombinant GHU protein. Based on the amount of GHU protein purified per 100 mL of induction culture medium, the 2×TY-Soya medium yielded the highest protein expression level among the seven media, approximately 2.15 mg / 100 mL, significantly higher than the other groups. The 2×TY and 2×TY-Casein media followed, with protein yields of approximately 1.88 mg / 100 mL and 1.78 mg / 100 mL, respectively. The LB-Soya medium also showed a certain improvement compared to the LB medium. In contrast, the protein expression levels in the 2×TY-Gelatin and 2×TY-Pea media were lower, both below the levels of the basal media. Comprehensive analysis indicates that changes in the type of nitrogen source and nutrient composition in the culture medium significantly affect the expression efficiency of GHU protein, with the 2×TY-Soya medium being the most favorable for GHU protein expression and serving as the optimal condition for subsequent culture medium optimization.

[0047] When the induction medium was 2×TY-Soya medium, the colorimetric results of determining the concentration of purified protein using the BCA method were as follows: Figure 3 As shown, Figure 3 The first column (wells 1A to 1H) contains standard protein solutions with concentrations ranging from 0.025 to 0.5 mg / mL, used to establish a standard curve. The remaining wells (columns 7 to 9, and 10 to 12) contain elution solutions of various gradients. These samples are from parallel protein purification experiments conducted with the same bacterial count. All samples were incubated at 60°C for 35 minutes before color development. Table 2 shows the protein concentrations (unit: mg / mL) calculated from the standard curves. The recombinant target protein with the His-tag was purified by nickel column affinity chromatography, and each fraction was analyzed by SDS-PAGE. Figure 4 The results show that Figure 4 In lanes B of the assay, lanes 4-10 all showed a major protein band slightly below 35 kDa, consistent with the theoretical molecular weight of the target protein (32.97 kDa). The target band was particularly prominent in lanes 5-7, indicating that this elution region was the main enriched component of the target protein. Besides the target band, only a few weak contaminant bands were observed, indicating that the target protein was significantly enriched after nickel column affinity purification, demonstrating good purification efficiency suitable for subsequent experiments. Based on the average protein concentration and the total volume of the protein solution (22 mL), the total expression level of the target protein was calculated to be 2.2 mg. This allows for the determination of the protein yield per unit of bacterial culture (100 mL), enabling the supplementation of amino acid substrates as needed in subsequent dipeptide synthesis experiments.

[0048] Table 2. Protein concentrations of each sample calculated based on the standard curve.

[0049] Example 2: Application of α-glutamate ligase GHU in the catalytic synthesis of proline-threonine dipeptides 1. Evaluate the substrate selectivity of α-glutamate ligase GHU Given that GHU protease was previously only a putative protein, its function was mainly inferred from sequence homology comparisons to be an enzyme of the RimK family from an unidentified species of α-proteobacteria: catalyzing the extension of glutamate residues at the C-terminus of proteins or peptides, and possibly participating in the synthesis of free dipeptides or small peptides. Therefore, we first provided a variety of monomeric amino acids as potential substrates to preliminarily investigate its catalytic synthesis ability and substrate adaptability.

[0050] After obtaining the relationship between bacterial culture and protein expression levels, expression was induced under the same conditions. 2 mM of 20 common amino acid substrates (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) were added to 2×TY-Soya expression bacterial culture (6 h after adding the inducer). The pH of the reaction solution was adjusted to 7.4, and the mixture was gently shaken at 35℃ for 16 hours. After the reaction, the mixture was sonicated for 30 min, centrifuged, and the supernatant was collected, lyophilized, and stored for later testing.

[0051] Table 3 presents the experimental results for evaluating the substrate selectivity of α-glutamate ligase GHU (upper performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q / Tof-MS) was used to identify dipeptide components in the samples). Given the complex composition of the sample matrix, to improve the reliability of the results interpretation, only signals with a response value > 10000 and a mass error < 5 mDa were considered valid detected products. The results showed that, in addition to added monomeric amino acids, several significantly generated dipeptides were detected, mainly including Pro-Thr, Ser-Cys, and His-His. The observed retention time (RT) of Ser-Cys was 0.57 min, significantly deviating from the typical retention time range of dipeptides, suggesting that this signal may be affected by matrix interference, co-elution, or misannotation. The detection of His-His may be related to a certain degree of self-polymerization / self-condensation tendency of His in the system, leading to the observation of this dipeptide product.

[0052] Table 3. Experimental results for evaluating the substrate selectivity of α-glutamate ligase GHU

[0053] 2. Synthesis of proline-threonine dipeptides using proline and threonine as substrates and catalyzed by α-glutamate ligase GHU. After analyzing the substrate profile of α-glutamate ligase GHU synthesis (Table 3), further verification experiments were conducted on the synthesis of proline-threonine (Pro-Thr). Specifically, 10 mM Pro and 10 mM Thr were added to 2×TY-Soya expression bacterial culture (expressed for 5 h after adding the inducer), the pH of the reaction solution was adjusted to 7.4, and the mixture was gently shaken at 35℃ for 16 hours. After the reaction, the mixture was sonicated for 30 min, centrifuged, and the supernatant was collected, lyophilized, and stored for further analysis. The dipeptide components of the samples were identified using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q / Tof-MS, Waters Xevo G3 Q Tof).

[0054] Figure 5 and Figure 6 The results of detecting the formation of proline-threonine dipeptide in the product under optimized liquid chromatography-mass spectrometry conditions are presented. Figure 5 As shown, in the liquid chromatography-mass spectrometry (LC-MS) results of the sample, a distinct chromatographic peak corresponding to the proline-threonine dipeptide Pro-Thr was detected at a retention time of approximately 3.47 min. The extracted ion mass-to-charge ratio corresponding to this peak is m / z 217.1190, which corresponds to the protonated molecular ion [M+H] of Pro-Thr. + The match indicates the presence of characteristic Pro-Thr chromatographic responses in the sample. Within the high-resolution mass spectrometry detection window of retention time 3.4699 ± 0.0192 min ( Figure 6 A major ion peak at m / z 217.11895 was detected in the low-energy channel, which corresponds to the fully protonated molecular ion [M+H] of the proline-threonine dipeptide Pro-Thr. + The signal matched; the corresponding ion signal at m / z 217.12087 was detected simultaneously in the high-energy channel, further supporting the existence of Pro-Thr related ion responses within this retention time window. Figure 6 The green-marked area shows the Pro-Thr target ion and its detected signals within its relevant mass range. The signal near m / z 218.12272 can be attributed to a naturally occurring isotope-related ion of Pro-Thr. Signals at m / z 219.17484 and m / z 220.07506, appearing simultaneously in the high-energy channel, can be considered as accompanying ion signals within this detection window, possibly originating from co-eluting compounds, substances of similar mass, or related ions formed under high-energy conditions. The results from both the low- and high-energy channels collectively indicate that Pro-Thr was effectively detected in the sample.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of α-glutamate ligase GHU in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides, characterized in that... The amino acid sequence of the α-glutamate ligase GHU is shown in SEQ ID NO.

1.

2. The use of the gene encoding the α-glutamate ligase GHU as described in claim 1 in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. The use of a recombinant expression vector containing any of the genes described in claim 2 or 3 in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

5. The application according to claim 4, characterized in that, The recombinant expression vector has a vector backbone of pET-21a(+).

6. The use of recombinant bacteria containing any of the genes described in claim 2 or 3 or any of the recombinant expression vectors described in claim 4 or 5 in the soybean protein-mediated catalytic synthesis of proline-threonine dipeptides.

7. The application according to claim 6, characterized in that, The host cell of the recombinant bacteria is Escherichia coli. C43 (DE3) .

8. A method for catalytic synthesis of proline-threonine dipeptides, characterized in that, Using proline and threonine as substrates, an enzymatic reaction is carried out in the presence of α-glutamate ligase GHU to generate a proline-threonine dipeptide; the amino acid sequence of the α-glutamate ligase GHU is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that, The enzymatic reaction conditions are pH 7.4, temperature 35℃, and reaction time 12-16 h.

10. The method according to claim 8, characterized in that, The α-glutamate ligase GHU was obtained by inoculating recombinant Escherichia coli overexpressing the α-glutamate ligase GHU gene into 2×TY-Soya medium and adding IPTG to induce expression; the nucleotide sequence of the α-glutamate ligase GHU gene is shown in SEQ ID NO.2; the 2×TY-Soya medium consists of 16 g / L soybean protein hydrolysate, 10 g / L yeast extract, 5 g / L sodium chloride, and the remainder is water.

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