Glutamate dehydrogenase mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms and enzyme engineering, in particular to a glutamate dehydrogenase mutant as well as a preparation method and application thereof. The mutant has specific amino acid mutation at the 5th site, the 20th site, the 44th site, the 298th site, the 313th site and the 443th site of a wild type, an amino acid sequence is shown as SEQ ID NO: 1, and a base sequence is shown as SEQ ID NO: 3 after a corresponding codon is optimized. According to the present invention, the efficient expression of the mutant is achieved through the recombinant expression vector pET24a-gdha.xl and the recombinant expression strain BL21 (DE3)-pET24a-gdha.xl; compared with a wild type, the mutant has the advantages that the enzyme activity is improved by about 4.5 times, the catalytic efficiency is obviously improved, and the activity is kept at high temperature and in a relatively wide pH range. The mutant can be used for catalyzing NADP < + > to be reduced into NADPH, cyclic utilization of NADPH is achieved, the industrial cost is reduced, and the mutant has remarkable application value.

Description

Technical Field

[0001] This invention relates to the fields of microbial and enzyme engineering technology, and in particular to a glutamate dehydrogenase mutant, its preparation method, and its application. Background Technology

[0002] Glutamate dehydrogenase (GDH, EC 1.4.1.4) is widely found in the mitochondria of prokaryotes and eukaryotes. This enzyme catalyzes the oxidative deamination of L-glutamate, while simultaneously converting the oxidized coenzyme NADP. + The reduced form of NADPH is one of the key enzymes in synthetic biology for achieving the NADPH cycle. Its catalytic reaction is as follows: L-glutamate + NADP + +H₂O→α-Ketoglutarate + NADPH + NH₄⁺ + +H + Among them: L-glutamic acid is oxidized to α-ketoglutarate, while NADP is... + It is reduced to NADPH. From a synthetic biology perspective, this reaction system has significant advantages: the substrate glutamate is inexpensive, while the byproducts α-ketoglutarate and ammonium salts are biocompatible, non-toxic, and do not inhibit coexisting enzymes, proteins, or other biomolecules. Therefore, the coenzyme regeneration system attached to glutamate dehydrogenase is widely used in biocatalytic processes in the food and pharmaceutical industries, such as the combined production of multiple dehydrogenases (e.g., the production of tetrahydrocurcumin in E. coli cells).

[0003] In industrial biocatalysis processes, NADPH is expensive and chemically unstable, making direct exogenous addition costly. A glutamate dehydrogenase-mediated coenzyme regeneration system, however, requires only a small initial amount of NADP. + In the presence of excess glutamate substrate, NADPH can be continuously generated through an enzymatic reaction. The generated NADPH is then used as a reducing agent in various other reactions to regenerate NADP. + It can re-enter the glutamate dehydrogenase catalytic cycle, thus forming a highly efficient and economical dynamic equilibrium system, significantly reducing the cost of coenzyme use. The core of the system's application efficiency depends on the catalytic efficiency and operational stability of the glutamate dehydrogenase itself.

[0004] However, the widely used wild-type glutamate dehydrogenase has significant limitations: its soluble expression level in heterologous expression systems (such as E. coli) is poor; at the same time, due to insufficient stability, it is prone to denaturation and inactivation under the temperature, pH, and other conditions required for industrial biocatalysis, resulting in low conversion rates of the final product and thus reducing the efficiency of the production process. Therefore, improving the soluble expression level and thermal stability of glutamate dehydrogenase has become crucial for promoting the industrial application of this technology.

[0005] To address the aforementioned issues, molecular modification of glutamate dehydrogenase is an effective solution. The aim is to use genetic engineering or protein engineering methods to directionally modify the amino acid sequence of glutamate dehydrogenase, such as mutations, insertions, deletions, and fusions, to improve its catalytic performance, stability, and soluble expression, thereby enhancing its adaptability to industrial applications. Summary of the Invention

[0006] In view of this, the present invention proposes a glutamate dehydrogenase mutant, its preparation method, and its application. The mutant is derived from *Escherichia coli* (…). Escherichia coli By modifying the wild-type glutamate dehydrogenase WtGdhA (K12), site-directed mutations were performed at six sites in its amino acid sequence to obtain a glutamate dehydrogenase mutant GdhA.XL with significantly improved catalytic activity, thermal stability, and pH stability. This mutant can efficiently and economically catalyze the regeneration of the coenzyme NADPH, making it suitable for industrial applications and overcoming the shortcomings of low catalytic efficiency and insufficient stability of wild-type glutamate dehydrogenase in the existing technology.

[0007] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a glutamate dehydrogenase mutant, which is obtained by point mutation of amino acids based on the amino acid sequence of wild-type glutamate dehydrogenase in Escherichia coli. The point mutations include: tyrosine at position 5 being mutated to cysteine ​​(Y5C), asparagine at position 20 being mutated to histidine (N20H), lysine at position 44 being mutated to arginine (K44R), lysine at position 298 being mutated to arginine (K298R), leucine at position 313 being mutated to valine (L313V), and alanine at position 443 being mutated to serine (A443S).

[0008] Based on the above technical solutions, preferably, the amino acid sequence of the glutamate dehydrogenase mutant is shown in SEQ ID NO: 1.

[0009] The sequence SEQ ID NO: 1 is as follows: MDQTCSLESFLNHVQKRDPHQTEFAQAVREVMTTLWPFLEQNPRYRQMSLLERLVEPERVIQFRVVWVDDRNQIQVNRAWRVQFSSAIGPYKGGMRFHPSVNLSILKFLGFE QTFKNALTLPMGGGKGGSDFDPKGKSEGEVMRFCQALMTELYRHLGADTDVPAGDIGVGGREVGFMAGMMKKLSNNTACVFTGKGLSFGGSLIRPEATGYGLVYFTEAMLK RHGMGFEGMRVSVSGSGNVAQYAIEKAMEFGARVITASDSSGTVVDESGFTKEKLARLIEIKASRDGRVADYAREFGLVYLEGQQPWSVPVDIALPCATQNELDVDAAHQLI ANGVKAVAEGANMPTTIEATELFQQAGVLFAPGKAANAGGVATSGLEMAQNAARLGWKAEKVDARLHHIMLDIHHACVEHGGEGEQTNYVQGANIAGFVKVADAMLSQGVI.

[0010] Based on the above technical solutions, preferably, the wild-type glutamate dehydrogenase of *Escherichia coli* is derived from *Escherichia coli* (…). Escherichia coli The wild-type glutamate dehydrogenase WtGdhA (K12) has the amino acid sequence shown in SEQ ID NO: 2, and UniProt id is P0037.

[0011] The sequence SEQ ID NO:2 is as follows: MDQTYSLESFLNHVQKRDPNQTEFAQAVREVMTTLWPFLEQNPKYRQMSLLERLVEPERVIQFRVVWVDDRNQIQVNRAWRVQFSSAIGPYKGGMRFHPSVNLSILKFLGFE QTFKNALTLPMGGGKGGSDFDPKGKSEGEVMRFCQALMTELYRHLGADTDVPAGDIGVGGREVGFMAGMMKKLSNNTACVFTGKGLSFGGSLIRPEATGYGLVYFTEAMLK RHGMGFEGMRVSVSGSGNVAQYAIEKAMEFGARVITASDSSGTVVDESGFTKEKLARLIEIKASRDGRVADYAKEFGLVYLEGQQPWSLPVDIALPCATQNELDVDAAHQLI ANGVKAVAEGANMPTTIEATELFQQAGVLFAPGKAANAGGVATSGLEMAQNAARLGWKAEKVDARLHHIMLDIHHACVEHGGEGEQTNYVQGANIAGFVKVADAMLAQGVI.

[0012] In a second aspect, a gene encoding a glutamate dehydrogenase mutant as described above is provided, the base sequence of which is shown in SEQ ID NO: 3.

[0013] The base sequence corresponding to the modified glutamate dehydrogenase mutant GdhA.XL was codon optimized to obtain the base sequence gdha.xl of the glutamate dehydrogenase mutant, as shown in SEQ ID NO: 3.

[0014] The sequence SEQ ID NO:3 is as follows:

[0015] The base sequence corresponding to the wild-type glutamate dehydrogenase WtGdhA was codon-optimized to obtain the base sequence Wtgdha of the wild-type glutamate dehydrogenase, as shown in SEQ ID NO: 4. Base fragments of glutamate dehydrogenase mutants and wild-type glutamate dehydrogenase containing XhoⅠ and NdeⅠ restriction sites were artificially synthesized.

[0016] The sequence SEQ ID NO:4 is as follows:

[0017] Thirdly, a recombinant expression vector is provided, which contains the gene of the glutamate dehydrogenase mutant as described above.

[0018] Fourthly, a recombinant strain is provided, which comprises the recombinant expression vector as described above.

[0019] Fifthly, a method for preparing the glutamate dehydrogenase mutant as described above is provided, comprising the following steps: S1, amplify the mutant gene of glutamate dehydrogenase; S2, construct a recombinant expression vector by inserting the glutamate dehydrogenase mutant gene into the empty vector to obtain the recombinant expression vector; S3, construct recombinant strains by transforming the recombinant expression vector into host bacteria to obtain recombinant strains; S4, Expression and purification of glutamate dehydrogenase mutant: After activation and expansion culture of recombinant strain, expression of glutamate dehydrogenase mutant was induced. Crude enzyme solution was obtained by cell disruption, followed by purification and concentration to obtain purified protein of glutamate dehydrogenase mutant.

[0020] Based on the above technical solutions, preferably, in step S1, the primer sequences for amplifying the glutamate dehydrogenase mutant gene are as shown in SEQ ID NO: 5 (ATATACATATGGATCAGACCTGCAGCCTGGAA) and SEQ ID NO: 6 (GTGGTGCTCGAGAATCACGCCCTGGCTCAGCAT).

[0021] Based on the above technical solutions, preferably, in step S2, the empty vector includes pET24a; and in step S3, the host bacterium includes Escherichia coli BL21.

[0022] Based on the above technical solution, in a further optimized step S2, a synthetically produced glutamate dehydrogenase mutant gene fragment containing XhoⅠ and NdeⅠ restriction sites is inserted into the expression vector pET24a to obtain the recombinant expression vector pET24a-gdha.xl containing the glutamate dehydrogenase mutant fragment. The construction method of the control recombinant expression vector pET24a-wtgdha.xl is the same as above.

[0023] Based on the above technical solution, in a further preferred embodiment, in step S3, the recombinant expression vector pET24a-gdha.xl is introduced into the host strain *Escherichia coli* BL21(DE3) using electroporation; the recombinant expression vector pET24a-gdha.xl contains a glutamate dehydrogenase mutant gene fragment. The construction method of the control recombinant expression strain BL21(DE3)-pET24a-wtgdha.xl is the same as above.

[0024] Based on the above technical solution, a further optimized step S4 involves activating the recombinant strain BL21(DE3)-pET24a-gdha.xl, followed by large-scale culture. IPTG is then used to induce expression of the glutamate dehydrogenase mutant gene, and the culture continues to obtain the fermentation broth. The bacterial cells are then broken up, and the purified glutamate dehydrogenase mutant protein is finally obtained by Ni-NTA affinity chromatography and ultrafiltration concentration. The purified protein of the control wild-type glutamate dehydrogenase is obtained using the same method.

[0025] The enzymatic properties of the purified glutamate dehydrogenase mutant GdhA.XL were characterized. The results showed that the optimal pH of the mutant was 8.5 and the optimal temperature was 55℃, which were consistent with the wild-type glutamate dehydrogenase WtGdhA. Compared with the wild type, the mutant GdhA.XL of the present invention showed significant improvements in both thermal stability and pH stability, which facilitates its storage and application under a wider range of process conditions.

[0026] Sixthly, an application of the glutamate dehydrogenase mutant described above in the catalytic regeneration of coenzyme NADPH is provided.

[0027] Based on the above technical solutions, preferably, the pH range of the catalytic reaction system is 8.0~9.5 and the temperature is 40~60℃.

[0028] Based on the above technical solution, the final concentrations of each compound in the catalytic reaction system are further optimized as follows: 100 mM phosphate buffer (pH 8.5), 5 mM L-glutamic acid, and 1 mM NADP. + Make up the volume to 1 mL with ultrapure water, mix thoroughly at 55℃, and then add glutamate dehydrogenase mutant to a final concentration of 20 μg / mL. This glutamate dehydrogenase mutant uses glutamate as a substrate to catalyze NADP. + When reduced to NADPH, at a pH of 8.5 and a temperature of 55°C, the enzyme activity of the glutamate dehydrogenase mutant can reach approximately 101.9 U / mg.

[0029] Glutamate dehydrogenase mutants can efficiently catalyze the following reaction: L-glutamate + NADP + +H₂O→α-Ketoglutarate + NADPH + NH₄⁺ + +H + .

[0030] In a seventh aspect, an enzyme preparation for the regeneration of coenzyme NADPH is provided, comprising the glutamate dehydrogenase mutant as described above.

[0031] The glutamate dehydrogenase mutant GdhA.XL of the present invention has the following advantages over the prior art: 1. At the optimal pH of 8.5 and the optimal temperature of 55℃, the enzyme activity of the glutamate dehydrogenase mutant reached approximately 101.9 U / mg. This represents an increase of nearly 4.5 times compared to the wild-type glutamate dehydrogenase activity of 22.8 U / mg, demonstrating superior enzyme activity. The glutamate dehydrogenase mutant can achieve stable recycling of NADPH in a coenzyme regeneration system, enabling efficient and continuous NADPH production and significantly reducing industrial costs.

[0032] 2. The catalytic constant of the glutamate dehydrogenase mutant at the optimal pH of 8.5 and the optimal temperature of 55℃. k cat It is 76.4 s -1 Compared to wild-type glutamate dehydrogenase k cat 17.1 s -1 The Michaelis constant for glutamate was increased by approximately 4.4 times; K M It is 0.65 mM, compared to wild-type glutamate dehydrogenase. K M The concentration was reduced by approximately 50% to 1.30 mM, indicating that the modified enzyme has a higher affinity for the substrate glutamate. The catalytic efficiency of the glutamate dehydrogenase mutant... k cat / K M It is 1.17 × 10 5 M -1 s -1 Compared to wild-type glutamate dehydrogenase (1.32 × 10⁻⁶), 4 M -1 s -1 The efficiency was increased by about 9 times, indicating that the modified glutamate dehydrogenase has a significant improvement in catalytic efficiency.

[0033] 3. In the thermostability test, the wild-type glutamate dehydrogenase showed a significant decrease in activity of only about 30% after incubation at 65℃ for 30 minutes, and almost no residual activity after incubation at 70℃. The glutamate dehydrogenase mutant exhibited higher residual activity at the same temperature, especially after incubation at 60℃ and 65℃ for 30 minutes, maintaining approximately 85% and 70% of the activity, respectively; and after incubation at 30℃, 35℃, and 40℃, maintaining approximately 80%, 85%, and 90% of the activity, respectively; indicating that the glutamate dehydrogenase mutant has superior thermostability compared to the wild type.

[0034] 4. In the stability study under different pH conditions, after incubation for 30 minutes in pH 9 buffer, the glutamate dehydrogenase mutant retained approximately 85% of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 55%. After incubation for 30 minutes in pH 10 buffer, the glutamate dehydrogenase mutant retained approximately 65% ​​of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 10%. In a slightly acidic environment, after incubation for 30 minutes in pH 6 buffer, the glutamate dehydrogenase mutant retained approximately 80% of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 60%. In a neutral environment, after incubation for 30 minutes in pH 7 buffer, the glutamate dehydrogenase mutant retained approximately 90% of its enzyme activity, and the wild-type glutamate dehydrogenase retained approximately 85%. This demonstrates that the glutamate dehydrogenase mutant exhibits good pH stability.

[0035] 5. The glutamate dehydrogenase mutant GdhA.XL exhibits high catalytic activity, high catalytic efficiency, and good thermal and pH stability. Therefore, only a small amount of NADP is required. + Initiating the reaction, in the presence of excess glutamate substrate, the glutamate dehydrogenase mutant can reduce NADP. + The process generates NADPH, which is used to oxidize L-glutamic acid to produce α-ketoglutarate. α-ketoglutarate has good biocompatibility and is harmless to the environment. The generated NADPH, as a reducing agent, is used in various other reactions to regenerate NADP. + These NADPs + It can re-enter the reaction system of glutamate dehydrogenase. Due to the excess of the substrate glutamate, the reaction will continue to form a dynamic equilibrium, stably and continuously producing NADPH. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a design diagram of the glutamate dehydrogenase mutation site of the present invention; Figure 2 This is a comparison diagram of the amino acid sequences of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase of the present invention; Figure 3This is a schematic diagram illustrating the construction of the expression vector pET24a-gdha.xl containing the glutamate dehydrogenase mutant gene and the expression vector pET24a-wtgdha containing the wild-type glutamate dehydrogenase gene of the present invention. Figure 4 This is a gel image of the glutamate dehydrogenase mutant in Example 2 of the present invention; Figure 5 This is a gel image of wild-type glutamate dehydrogenase in Example 2 of the present invention; Figure 6 This is a bar chart comparing the enzyme activity of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase in Example 3 of the present invention. Figure 7 This is a bar chart comparing the thermostability of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase in Example 5 of the present invention; Figure 8 This is a bar chart comparing the pH stability of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase in Example 6 of the present invention. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Construction of glutamate dehydrogenase mutant.

[0040] 1. Obtain the amino acid sequence of the glutamate dehydrogenase mutant.

[0041] Based on wild type Escherichia coli The amino acid sequence of glutamate dehydrogenase from strain K12, serial number P00370 in the uniprot database, is shown in SEQ ID NO: 2. Site-directed mutagenesis was then performed at relevant sites through a rational design, such as... Figure 1 As shown.

[0042] (1) The tyrosine at position 5 is mutated to cysteine ​​Y5C. This position is located on the protein surface. By mutating the relatively hydrophobic tyrosine to cysteine, the surface hydrophobicity can be reduced and the hydrophilicity of the protein surface can be enhanced. At the same time, in the redox reaction catalyzed by this enzyme, the electron transfer rate of the thiol group on the side chain of cysteine ​​as an electron transfer station is better than that of the phenolic hydroxyl group of tyrosine.

[0043] (2) The asparagine at position 20 is mutated to histidine N20H, introducing a positively charged group at this position, which enhances the hydrophilic interaction with the solvent and can significantly improve the thermal stability, chemical stability and solubility of the protein. At the same time, by stabilizing the folding intermediate / transition state, reducing misfolding pathways and promoting hydrophobic collapse, it can also effectively improve the folding efficiency and yield of the protein.

[0044] (3) The 44th lysine is mutated to arginine K44R. The guanidinium group of the arginine side chain forms a local hydrogen bond network with the tyrosine at position 45 and the aspartic acid at position 439, which can effectively improve the thermal stability of the enzyme molecule and the folding efficiency during the expression process.

[0045] (4) The lysine at position 298 is mutated to arginine K298R. Similar to K44R, the guanidinium group of the newly introduced arginine side chain forms a hydrogen bond with the carboxyl group on the aspartic acid side chain at position 295, which can effectively improve the thermal stability of the enzyme molecule and the folding efficiency during the expression process.

[0046] (5) The leucine at position 313 is mutated to valine L313V, which reduces the volume of the side chain group, thereby reducing steric conflict, optimizing van der Waals contact distance, lowering local energy, and thus reducing the protein folding conformational entropy loss. At the same time, the valine side chain is smaller and more flexible, which can promote the early formation of the hydrophobic core in this region during protein expression and shorten the time of the initial folding step.

[0047] (6) The alanine at position 443 is mutated to serine A443S, which forms a hydrogen bond with the aspartic acid backbone at position 439 on the α-helix. This can effectively improve the thermal stability of the enzyme molecule.

[0048] The amino acid sequence of the glutamate dehydrogenase mutant GdhA.XL was thus obtained, as shown in SEQ ID NO: 1. The amino acid sequence of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase are compared as follows: Figure 2 As shown.

[0049] 2. Obtaining the base sequence of the glutamate dehydrogenase mutant.

[0050] Based on the amino acid sequence of the glutamate dehydrogenase mutant GdhA.XL, the base sequence of the glutamate dehydrogenase mutant, gdhA.xl, was artificially redesigned. The specific method is as follows: Since the amino acid sequence is encoded by codons on mRNA, and the mRNA sequence is complementary to the DNA template strand, the possible mRNA sequence can be deduced from the codons of the amino acid sequence, and further, the DNA base sequence can be deduced. However, due to the degeneracy of codons, there are multiple possible DNA base sequences. Therefore, high-frequency codons from *E. coli* were used to replace low-frequency codons, optimizing the base sequence of the glutamate dehydrogenase mutant. The base sequence of the glutamate dehydrogenase mutant gene gdhA.xl is shown in SEQ ID NO: 3. Similarly, based on the amino acid sequence WtGdhA of wild-type glutamate dehydrogenase, the base sequence of wild-type glutamate dehydrogenase was artificially redesigned, using high-frequency codons from *E. coli* to replace low-frequency codons. The codon-optimized base sequence of wild-type glutamate dehydrogenase, wtgdhA, is shown in SEQ ID NO: 4. After the above optimizations, the proportion of high-frequency codons (such as GAA, GGC, CTG, and GCT) in the gene was significantly increased, effectively improving translation efficiency and protein expression levels. This optimization strategy not only enhanced expression stability but also reduced the risk of protein misfolding and inclusion body formation, providing a theoretical basis for the subsequent efficient expression of the glutamate dehydrogenase mutant GdhA.XL and the wild-type glutamate dehydrogenase WtGdhA.

[0051] 3. Obtaining the glutamate dehydrogenase mutant gene fragment and the expression vector containing the glutamate dehydrogenase mutant gene fragment.

[0052] The gene sequence of the glutamate dehydrogenase mutant, as shown in SEQ ID NO: 3, and the gene sequence of the wild-type glutamate dehydrogenase, as shown in SEQ ID NO: 4, were synthesized by a gene synthesis company. The glutamate dehydrogenase mutant gene sequence was amplified by PCR, and both ends of the gene contained XhoⅠ and NdeⅠ restriction sites. The primer for the glutamate dehydrogenase mutant was ATATA. CATA TG GATCAGACCTGCAGCCTGGAA (SEQ ID NO: 5), GTGGTG CTCGAG AATCACGCCCTGGCTCAGCAT (SEQ ID NO: 6); the primer for wild-type glutamate dehydrogenase is ATATA. CATATG GATCAGACCTATAGCCTGGAA,GTGGTG CT CGAGAATCACGCCCTGCGCCAGCATCGCA. The mutant glutamate dehydrogenase was ligated into the commonly used expression vector pET24a using a double enzyme digestion technique, resulting in the recombinant expression vector pET24a-gdha.xl for glutamate dehydrogenase and the control wild-type glutamate dehydrogenase expression vector pET24a-wtgdha.

[0053] The schematic diagrams of the construction process of the glutamate dehydrogenase mutant recombinant expression vector pET24a-gdha.xl and the control wild-type glutamate dehydrogenase expression vector pET24a-wtgdha are shown below. Figure 3 As shown.

[0054] Example 2: Obtaining and expressing the recombinant expression strain BL21(DE3)-pET24a-gdha.xl of glutamate dehydrogenase mutant.

[0055] 1. Electroporation: Mix 5 μL of the recombinant expression vector pET24a-gdha.xl with 50 μL of *E. coli* BL21(DE3) competent cells and incubate on ice for 5 min. Using a pre-chilled electroporation cuvette, set the electroporator parameters (voltage 1800V, resistance 200Ω, capacitance 25μF) and electroporate the mixture of recombinant vector and competent cells on ice. Immediately add 1 mL of preheated LB medium (37 ℃), transfer to a 1.5 mL centrifuge tube, and incubate at 37 ℃ for 2 hours. Spread 200 μL of the bacterial culture onto LB agar plates containing kanamycin and chloramphenicol, and incubate at 37 ℃ for 24 hours. After single colonies have grown, the recombinant positive strain of *E. coli* containing the gdha.xl gene is obtained. The construction method of the control wild-type glutamate dehydrogenase recombinant expression strain BL21(DE3)-pET24a-wtgdha is the same as above.

[0056] 2. Protein induction and lysis: The recombinant strain was inoculated into 5 mL of LB liquid medium and cultured overnight at 37°C until OD200 reached. 600 The expression level was 3.0-6.0. The cells were transferred to 250 mL LB liquid medium and cultured at 37°C for 8 h. IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C for 12-16 h to obtain the fermentation broth. The cells were collected by centrifugation at 8000 rpm for 15 min, resuspended in 50 mM pH 7.0 phosphate buffer, and disrupted by high-pressure grinding. The cells were then centrifuged again at 8000 rpm for 15 min, and the supernatant was collected. The induction and disruption methods for the control wild-type glutamate dehydrogenase protein were the same.

[0057] 3. Ni-NTA affinity chromatography purification: Bacterial cell lysates containing the glutamate dehydrogenase mutant were loaded onto a Ni-NTA column, and the flow-through (FT) was collected. The cells were washed three times with lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0, and filtered sterile). The cells were then washed once with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20-50 mM imidazole, pH 8.0, and filtered sterile), and the washings were collected. The cells were eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250-500 mM imidazole, pH 8.0, and filtered sterile), and 12 tubes (1 mL each) of eluent were collected. Coomassie brilliant blue staining was used to identify the protein bands. The purification method for the wild-type glutamate dehydrogenase control was the same. Results are as follows: Figure 4 and Figure 5 As shown, SDS-PAGE results indicate that both strains successfully expressed the target protein, and high-purity products were obtained after Ni-NTA purification. Figure 4 Elutions 1 through 7 are the target proteins of glutamate dehydrogenase mutants eluted in multiple small-volume elutions. Figure 5 Elutions 1 through 7 are wild-type glutamate dehydrogenase target proteins eluted in multiple small volumes.

[0058] 4. Protein Concentration and Dialysis: The eluents containing the glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase were dialyzed, and then concentrated using ultrafiltration tubes. The eluents were centrifuged at 5000 rpm to 1.5 mL and stored.

[0059] Example 3: Determination of the activity of glutamate dehydrogenase mutant.

[0060] 1. Protein concentration determination of glutamate dehydrogenase mutants (BCA method): Preparation of BCA working solution: Mix 50 volumes of solution A with 1 volume of solution B; Construction of standard curve: Take 0-20 μL of standard protein (to achieve a protein concentration of 0-200 μg / mL), add 200 μL of BCA working solution, react at 60℃ for 30 minutes, and measure the absorbance at A562. The BCA method is the dioctoctanic acid method, the principle of which is: under alkaline conditions, protein will convert Cu... 2+ Reduced to Cu + Cu +It then forms a purple complex with BCA (dioctoctanic acid), and its absorbance at 562 nm is directly proportional to the protein concentration. A is the alkaline buffer system for dioctoctanic acid (BCA), and B is a 4% copper sulfate pentahydrate (CuSO4·5H2O) solution. Sample determination: Following the same procedure, an appropriate volume of sample was added, followed by water to a final volume of 20 μL. Then, 200 μL of BCA working solution was added to the reaction system. The reaction was carried out in an oven at 60 °C for 30 min, and the absorbance at A562 nm was measured. Finally, the protein concentration was calculated based on the standard curve. Using the above method, the protein concentration of the glutamate dehydrogenase mutant obtained in Example 2 was determined to be 4.3 mg / mL, and the protein concentration of the wild-type glutamate dehydrogenase was 2.06 mg / mL.

[0061] 2. Definition of enzyme activity of glutamate dehydrogenase.

[0062] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 µmol of NADPH per minute under specific conditions (e.g., 37°C, pH 8.5), expressed in U / mg.

[0063] L-glutamate + NADP + +H₂O→α-Ketoglutarate + NADPH + NH₄⁺ + +H + The change in absorbance of A (340 nm) was measured using a spectrophotometer at room temperature. Under standard conditions, the reaction mixture was prepared as shown above, with a final volume of 1 mL. The reaction rate was recorded. The enzyme activity was calculated using the following formula:

[0064] in, : NADP was measured at A (340 nm) + Changes in absorbance; T The measurement time interval is 5 minutes. ε The molar extinction coefficient of NADPH is 6220 M. -1 cm -1 ; l The optical path length of the cuvette is 1 cm; E Protein concentration, µg / mL; V 酶 Enzyme volume, mL; V 反应体系 : The volume of the entire reaction system, mL.

[0065] 3. Catalytic activity of glutamate dehydrogenase mutants under different pH conditions.

[0066] Prepare a phosphate buffer solution with a pH gradient range of 7-9.5. The initial phosphate buffer concentration is 100 mM, the final concentration of L-glutamate is 5 mM, and the final concentration of NADP is 1 mM. + Take 50 μL of enzyme solution and make up the total volume to 1 mL with ultrapure water; react at 37℃ for 5 min. After the reaction is complete, measure the change in absorbance at A (340 nm) using a spectrophotometer at room temperature.

[0067] The highest enzyme activities of both the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase occurred at pH 8.5 at a temperature of 37℃. Therefore, the enzyme activity at pH 8.5 was normalized as 100% to examine the relative enzyme activity (%) at different pH values. Figure 6 As shown. According to Figure 6 The results showed that the glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase had the highest enzyme activity at pH 8.5, and the optimal pH was 8.5.

[0068] 4. Investigation into the optimal temperature for the glutamate dehydrogenase-catalyzed reaction.

[0069] Prepare a phosphate buffer solution with a pH of 8.5 and a concentration of 100 mM. The final concentration of L-glutamate is 5 mM, and the final concentration of NADP is 1 mM. + Take 50 μL of enzyme solution and make up the total volume to 1 mL with ultrapure water; react for 5 min in a temperature gradient range of 30~65℃. After the reaction is complete, measure the change in absorbance A (340 nm) at room temperature using a spectrophotometer.

[0070] At pH 8.5, the highest enzyme activities of both the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase occurred at a temperature of 55℃; therefore, the enzyme activity at 55℃ was normalized to 100% baseline to examine the relative enzyme activities (%) of the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase at different temperatures. Figure 7 As shown. According to Figure 7 The results showed that the optimal temperature for both the glutamate dehydrogenase mutant and the wild-type glutamate dehydrogenase was 55℃, at which the enzyme activity was the highest. Therefore, the optimal temperature was 55℃.

[0071] Therefore, the optimal temperature for obtaining the enzyme activity of the glutamate dehydrogenase mutant and the wild-type glutamate was 55℃, and the optimal pH was 8.5.

[0072] 5. Glutamate dehydrogenase mutant activity assay.

[0073] Prepare a 100 mM phosphate buffer (pH 8.5), a final concentration of 5 mM L-glutamate, and a final concentration of 1 mM NADP. +Add 50 μL of enzyme solution and make up the total volume to 1 mL with ultrapure water; react at 55 °C for 5 min. After the reaction is complete, measure the change in absorbance at A (340 nm) at room temperature using a spectrophotometer.

[0074] The enzyme activity of wild-type glutamate dehydrogenase was determined using the same method as above. Using the above method, at the optimal pH of 8.5 and the optimal temperature of 55℃, the enzyme activity of the glutamate dehydrogenase mutant was 101.9 U / mg, and the enzyme activity of the wild-type glutamate dehydrogenase was 22.8 U / mg. The bar charts for the enzyme activities of the glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase are shown below. Figure 8 As shown.

[0075] Example 4: Kinetic analysis of glutamate dehydrogenase mutants.

[0076] Kinetic analysis of the modified glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase was performed using conventional testing and analysis methods. The results are shown in Table 1.

[0077] Table 1. Kinetic constants and enzyme activities of glutamate dehydrogenase mutants

[0078] Based on the enzyme kinetic results shown in Table 1, the modified glutamate dehydrogenase mutant, compared with wild-type glutamate dehydrogenase, exhibits the following kinetic characteristics when catalyzing L-glutamate at optimal pH and temperature: 1. Catalytic constant: The glutamate dehydrogenase mutant... k cat It is 76.4 s -1 Compared to the 17.1 s of wild-type glutamate dehydrogenase... -1 It increased by approximately 4.4 times; 2. Michaelis constant: glutamate dehydrogenase mutant K M The concentration was 0.65 mM, a decrease of approximately 50% compared to the 1.30 mM of wild-type glutamate dehydrogenase, indicating that the modified enzyme has a higher affinity for the substrate. 3. Catalytic efficiency: The glutamate dehydrogenase mutant... k cat / K M It is 1.17 × 10 5 M -1 s -1 Compared to wild-type glutamate dehydrogenase (1.32 x 10⁻⁶), 4 M -1 s -1The 8.9-fold increase indicates a significant improvement in catalytic efficiency by the glutamate dehydrogenase mutant. The glutamate dehydrogenase mutant of this invention exhibits significantly improved catalytic efficiency and enzyme activity compared to wild-type glutamate dehydrogenase, and also demonstrates a higher affinity for the substrate monosodium glutamate (MSG). This provides a highly efficient enzyme catalyst for industrial production and biotechnology applications.

[0079] Example 5: Thermal stability analysis of glutamate dehydrogenase mutant.

[0080] 1. Sample preparation: The glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase obtained in Example 2 were diluted to 1 mg / mL with 0.1M Tris-HCl buffer (pH 8.5) and dispensed into 200 μL tubes, with 3 replicates per group.

[0081] 2. Temperature incubation treatment: The temperature gradient is set to 30, 35, 40, 45, 50, 55, 60, 65, and 70°C; the enzyme solution is incubated in a water bath at each temperature for 30 minutes, and then immediately terminated by placing it in an ice bath.

[0082] 3. Activity Assay: Residual activity was determined under the optimal conditions (pH 8.5, 55°C) as described in Example 3. The activity of the group treated at the optimal temperature of 55°C was normalized to 100% baseline, and the relative activities under other temperature conditions were calculated. The results are as follows: Figure 7 As shown.

[0083] according to Figure 7 Temperature-dependent analysis revealed that the wild-type glutamate dehydrogenase exhibited a significant decrease in activity after incubation at 65°C, retaining only about 40% of its activity. Incubation at 70°C resulted in almost no residual activity, and at 30°C, 35°C, and 45°C, the activity was only about 45%, 65%, and 65%, respectively. The glutamate dehydrogenase mutant showed higher residual activity at the same temperature, particularly maintaining over 85% and 70% of its activity at 60°C and 65°C, respectively; and maintaining over 80%, 85%, and 90% of its activity at 30°C, 35°C, and 40°C, respectively. Therefore, the glutamate dehydrogenase mutant demonstrated good thermostability.

[0084] The above results indicate that the modified glutamate dehydrogenase mutant exhibits stronger heat tolerance under high-temperature incubation conditions. The modification significantly enhances its conformational stability and resistance to thermal denaturation, providing a good foundation for its application in high-temperature industrial enzyme catalytic reactions. At temperatures ranging from 30℃ to 50℃, the relative enzyme activity of the glutamate dehydrogenase mutant is also more than 80% of that at the optimal temperature of 55℃, providing a wider temperature range for the short-term preservation of glutamate dehydrogenase and offering more possibilities for temperature selection in various complex enzyme catalytic reactions.

[0085] Example 6: pH stability analysis of glutamate dehydrogenase mutant.

[0086] 1. Sample preparation: The purified glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase were placed in Tris-HCl buffer at pH 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and incubated at 55°C for 30 minutes. The incubation was immediately stopped by placing the sample in an ice bath. Each group had 3 replicates.

[0087] 2. Activity Assay: Residual activity was determined under the optimal conditions (pH 8.5, 55°C) as described in Example 3. The activity of the treatment group at the optimal pH of 8.5 was normalized to 100% baseline, and the relative activities at other pH values ​​were calculated. The results are as follows: Figure 8 As shown.

[0088] according to Figure 8 The experimental results show that the pH stability of the glutamate dehydrogenase mutant is significantly better than that of the wild-type glutamate dehydrogenase, especially in alkaline environments (pH 9-10). Specifically, after incubation for 30 minutes in pH 9 buffer, the glutamate dehydrogenase mutant retained approximately 85% of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 55%. After incubation for 30 minutes in pH 10 buffer, the glutamate dehydrogenase mutant retained approximately 65% ​​of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 10%. In an acidic environment, after incubation for 30 minutes in pH 6 buffer, the glutamate dehydrogenase mutant retained approximately 75% of its enzyme activity, while the wild-type glutamate dehydrogenase retained only about 60%. In a neutral environment, after incubation for 30 minutes in pH 7 buffer, the glutamate dehydrogenase mutant retained approximately 90% of its enzyme activity, and the wild-type glutamate dehydrogenase retained approximately 85%. like Figure 8 As shown, this indicates that the glutamate dehydrogenase mutant maintains higher activity over a wider pH range compared to the wild-type glutamate dehydrogenase, and its pH stability is superior to that of the wild-type. The glutamate dehydrogenase mutant exhibits relatively high enzyme activity between pH 6 and 10, providing a greater adaptability for industrial applications in complex environments and offering a wider pH range for the short-term preservation of glutamate dehydrogenase.

[0089] Example 7: Application of glutamate dehydrogenase mutant.

[0090] Preparation of enzyme catalyst: The final concentration of the glutamate dehydrogenase mutant protein in Example 2 was 20 μg / mL, and the enzyme activity was 101.9 U / mg; the control enzyme was wild-type glutamate dehydrogenase, with a final protein concentration of 20 μg / mL and an enzyme activity of 22.8 U / mg.

[0091] Preparation of reaction system 1: Prepare 100 μL of 1 M phosphate buffer (pH 8.5), 5 μL of 1 M glutamate, and 100 mM NADP. +Add 10 μL of ultrapure water to make up the volume to 1 mL, mix well at 55℃, and then add 10 μL of 2 mg / mL glutamate dehydrogenase mutant. Prepare reaction system 2: 100 μL of 1 M phosphate buffer (pH 8.5), 5 μL of 1 M glutamate, and 100 mM NADP. + Add 10 μL of ultrapure water to make up the volume to 1 mL, mix well at 55℃, and then add 10 μL of wild-type glutamate dehydrogenase at a concentration of 2 mg / mL.

[0092] The comparison results of the application of glutamate dehydrogenase mutant and wild-type glutamate dehydrogenase in the production of NADPH are shown in Table 2.

[0093] Table 2. Comparison of NADPH production applications between glutamate dehydrogenase mutants and wild-type glutamate dehydrogenases.

[0094] As shown in Table 2, the glutamate dehydrogenase mutant produces nearly 5 times more NADPH within 5 minutes than the wild-type glutamate dehydrogenase under standard reaction conditions, providing a basis for the industrial application of the glutamate dehydrogenase mutant to mass-produce NADPH.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A glutamate dehydrogenase mutant, characterized in that: The glutamate dehydrogenase mutant was obtained by point mutation of amino acids based on the amino acid sequence of wild-type glutamate dehydrogenase in Escherichia coli. The point mutations included: tyrosine at position 5 was mutated to cysteine, asparagine at position 20 was mutated to histidine, lysine at position 44 was mutated to arginine, lysine at position 298 was mutated to arginine, leucine at position 313 was mutated to valine, and alanine at position 443 was mutated to serine.

2. The glutamate dehydrogenase mutant as described in claim 1, characterized in that: The amino acid sequence of the glutamate dehydrogenase mutant is shown in SEQ ID NO:

1.

3. A gene encoding a glutamate dehydrogenase mutant as described in claim 1 or 2, characterized in that: The base sequence of the glutamate dehydrogenase mutant is shown in SEQ ID NO:

3.

4. A recombinant expression vector, characterized in that: A gene containing the glutamate dehydrogenase mutant of claim 3.

5. A recombinant bacterial strain, characterized in that: It includes the recombinant expression vector as described in claim 4.

6. A method for preparing a glutamate dehydrogenase mutant as described in claim 1 or 2, characterized in that, Includes the following steps: S1, amplify the mutant gene of glutamate dehydrogenase; S2, construct a recombinant expression vector by inserting the glutamate dehydrogenase mutant gene into the empty vector to obtain the recombinant expression vector; S3, construct recombinant strains by transforming the recombinant expression vector into host bacteria to obtain recombinant strains; S4, Expression and purification of glutamate dehydrogenase mutant: After activation and expansion culture of recombinant strain, expression of glutamate dehydrogenase mutant was induced. Crude enzyme solution was obtained by cell disruption, followed by purification and concentration to obtain purified protein of glutamate dehydrogenase mutant.

7. A method for preparing the glutamate dehydrogenase mutant as described in claim 6, characterized in that: In step S1, the primer sequences for amplifying the glutamate dehydrogenase mutant gene are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

8. A method for preparing the glutamate dehydrogenase mutant as described in claim 6, characterized in that: In step S2, the empty vector includes pET24a; in step S3, the host bacterium includes Escherichia coli BL21.

9. The application of a glutamate dehydrogenase mutant as described in claim 1 or 2 in the catalytic regeneration of coenzyme NADPH.

10. An enzyme preparation for the regeneration of coenzyme NADPH, characterized in that: It includes the glutamate dehydrogenase mutant as described in claim 1 or 2.

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