L-amino acid deaminase mutant and application thereof

Abstract

The invention discloses an L-amino acid deaminase mutant and application thereof. The invention verifies that the L-amino acid deaminase derived from proteus haunhai has the capability of catalyzing L-glutamic acid to generate alpha-KG for the first time. Through protein three-dimensional structure simulation and the like, it is predicted that related sites possibly influencing catalysis and combination are I64, G257, Q278 and M440, site-saturated mutation is carried out, and a mutant and engineering bacteria for efficient catalytic synthesis of alpha-KG are obtained. Compared with a wild type, the catalytic activity of the mutant for catalyzing L-glutamic acid to generate alpha-ketoglutaric acid is remarkably improved, and the mutant has a wide prospect of large-scale industrial application.

Description

[0001] This application is a divisional application of Chinese invention patent application number "202510205986.4" entitled "An L-amino acid deaminase and its mutant and application". Technical Field

[0002] This invention relates to the field of biotechnology, specifically to a... Proteus hauseri L-amino acid deaminase mutants and their applications. Background Technology

[0003] α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle, participating in the synthesis of amino acids, vitamins, and organic acids, as well as energy metabolism in organisms. α-Ketoglutarate undergoes transamination to form L-glutamate, which enters nitrogen metabolism, serving as a key node connecting intracellular carbon and nitrogen metabolism. Common α-ketoglutarate derivatives include γ-aminobutyric acid (GABA), 5-aminolevulinic acid (5-ALA), and hydroxylated compounds such as 4-hydroxyisoleucine (4-HIL) catalyzed by α-ketoglutarate-dependent hydroxylases. To date, α-ketoglutarate and its derivatives have been widely used in numerous fields, including chemical, food, pharmaceutical, and daily chemical industries, demonstrating a very broad application prospect.

[0004] Currently, the main methods for synthesizing α-ketoglutarate (α-KG) include chemical methods, microbial fermentation methods, and enzymatic methods. Chemical methods typically use diethyl succinate and diethyl oxalate as raw materials, involving condensation, hydrolysis, distillation, concentration, and crystallization to synthesize α-ketoglutarate, achieving a maximum yield of 75%. However, the chemical method for α-KG production has a unique starting material structure, contains toxic substances that pollute the environment, requires multiple reaction steps, has a low final yield, and poses serious safety concerns, limiting its application in the food and cosmetics industries. Microbial fermentation for α-KG production has made significant progress over the decades, but it also suffers from disadvantages such as scarce production strains, low yields of metabolites, difficulties in separation, and long production cycles, failing to meet the needs of large-scale industrial production. Enzymatic conversion methods offer advantages such as mild reaction conditions, a single product, simple extraction processes, high product concentration, and high purification yield, making them the mainstream method for α-ketoglutarate production. There are three main pathways for the enzymatic synthesis of α-ketoglutarate, all using L-glutamic acid as a raw material. These pathways involve glutamated ehydrogenase (GDH), amino acid oxidase (AAO, EC 1.4.3.2 and EC 1.4.3.3), and L-amino acid deaminase (LAD, EC 1.4.3.2) to catalyze the synthesis of α-ketoglutarate.

[0005] The first pathway described above benefits from the fact that glutamate dehydrogenase has a higher affinity for α-ketoglutarate than glutamate, and this reaction requires the coenzyme nicotinamide adenine dinucleotide phosphate (NADP). + Because of the involvement of L-amino acid oxidase in pathway two, the yield of α-ketoglutarate is extremely low. L-amino acid oxidase in pathway two produces hydrogen peroxide during the reaction, severely inhibiting its activity. Although adding catalase (CAT) can eliminate hydrogen peroxide, its complex production process and increased production costs make it difficult to apply industrially. L-amino acid deaminase in pathway three also catalyzes the synthesis of α-ketoglutarate from L-glutamic acid. The reaction only produces α-ketoglutarate, ammonia, and water, without the production of toxic substances like H2O2, thus having less impact on cell growth and being more favorable for α-ketoglutarate production. Currently, research indicates that it originates from *Proteus mirabilis* (…). Proteus mirabilisThe L-amino acid deaminase (pm1) from *E. coli* BL21(DE3) can be heterologously expressed in the strain, effectively converting L-glutamate to α-ketoglutarate. However, as a membrane protein, pm1 has low expression levels and low specificity for the substrate L-glutamate (35.8%). Currently, research on L-amino acid deaminases is limited, and very few types of L-amino acid deaminases capable of effectively catalyzing the synthesis of α-ketoglutarate have been discovered; only those from [specific strain name missing] have been reported. Proteus mirabilis The presence of PM1 limits the industrial application of enzymatic synthesis of α-ketoglutarate. Therefore, discovering new, highly efficient L-amino acid deaminases and enhancing their activity through directed evolution will help further reduce the cost of industrial production. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing α-ketoglutaric acid synthesis processes, the primary objective of this invention is to provide a... Proteus hauseri L-amino acid deaminase mutant of the source.

[0007] Another object of the present invention is to provide the encoding gene of the above-mentioned L-amino acid deaminase mutant.

[0008] Another object of the present invention is to provide the application of the above-mentioned L-amino acid deaminase mutant in the biosynthesis of α-ketoglutarate.

[0009] This invention is the first to verify the presence of Proteus haematobacterium ( Proteus hauseri L-amino acid deaminases derived from [source name] have the ability to catalyze the synthesis of α-ketoglutarate from L-glutamate. Furthermore, through protein three-dimensional structure simulation, molecular docking, and virtual saturation mutagenesis, the relevant sites that may affect catalysis and binding were predicted to be ILE64, GLY257, GLN278, and MET440. Site-directed saturation mutagenesis was performed on these four sites to obtain mutants and engineered bacteria that efficiently catalyze the synthesis of α-ketoglutarate, showing promising application prospects.

[0010] The objective of this invention is achieved through the following technical solution: An L-amino acid deaminase mutant, whose amino acid sequence is SEQ ID NO: 1, is obtained by any of the following mutations: At least one of I64R, G257H, Q278R and M440K; wherein, I64R, i.e., the 64th amino acid, is mutated from ILE(I) to ARG(R), and the others are similar.

[0011] Furthermore, the L-amino acid deaminase mutant is obtained by any one of the following mutations, where the amino acid sequence is SEQ ID NO: 1: I64R, G257H, Q278R, M440K, I64R / M440K, Q278R / M440K, I64R / Q278R / M440K or I64R / G257H / Q278R / M440K.

[0012] Preferably, the gene sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0013] The encoding gene of the L-amino acid deaminase mutant.

[0014] Preferably, an L-amino acid deaminase mutant LAAD-phRK (I64R / G257H / Q278R / M440K) has the amino acid sequence shown in SEQ ID NO: 3.

[0015] The encoding gene of the L-amino acid deaminase mutant LAAD-phRK has the nucleotide sequence shown in SEQ ID NO: 4.

[0016] The aforementioned mutant-related biological materials are any one or more combinations of the following biological materials: (a) An expression cassette containing the above-mentioned encoded genes; (b) Recombinant expression vectors containing the above-mentioned coding genes; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant microorganisms containing the above-mentioned encoding genes; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

[0017] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a pET series vector or a pPICZα vector, etc.; preferably a pET-28a(+) vector, a pET-26b(+) vector or a pPICZαA vector.

[0018] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) are selected from prokaryotes or yeast, etc.; the prokaryotes include Escherichia spp. ( Escherichia Bacteria such as *Pichia pastoris*; the yeast includes *Pichia pastoris* and other yeasts. More specifically, the prokaryote is *Escherichia coli* (…). Escherichia coli , E. coli Specifically, it can be Escherichia coli BL21(DE3), Escherichia coli DH5α or Escherichia coli TOP10; the yeast is Pichia pastoris X33 or GS115.

[0019] The above-mentioned mutants, encoding genes, and mutant-related biological materials are used in the preparation of L-amino acid deaminase mutants.

[0020] Furthermore, the applications of mutants, coding genes, mutant-related biological materials, L-amino acid deaminase LAAD-ph3, and L-amino acid deaminase LAAD-ph3-related biological materials include one of the following applications: (I) Application in the biosynthesis of α-ketoglutaric acid; (II) Applications in food, pharmaceuticals, feed, cosmetics, and chemicals; (III) Application in health products.

[0021] The biomaterial related to the L-amino acid deaminase LAAD-ph3 is any one or more combinations of the following biomaterials: 1) An expression cassette containing the gene encoding the L-amino acid deaminase LAAD-ph3; 2) Recombinant expression vectors containing genes encoding the L-amino acid deaminase LAAD-ph3; 3) Recombinant expression vectors containing the expression cassette described in 1); 4) Recombinant microorganisms containing the gene encoding the L-amino acid deaminase LAAD-ph3; 5) Recombinant microorganisms containing the expression cassette described in 1); 6) Recombinant microorganisms containing the recombinant expression vector described in 2) or 3).

[0022] Furthermore, the starting vector for the recombinant expression vectors described in 2) and 3) is a pET series vector or a pPICZα vector, etc.; preferably, it is a pET-28a(+) vector, a pET-26b(+) vector or a pPICZαA vector.

[0023] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in 4), 5), and 6) are selected from prokaryotes or yeast, etc.; the prokaryotes include Escherichia spp. ( Escherichia Bacteria such as *Pichia pastoris*; the yeast includes *Pichia pastoris* and other yeasts. More specifically, the prokaryote is *Escherichia coli* (…). Escherichia coli , E. coli Specifically, it can be Escherichia coli BL21(DE3), Escherichia coli DH5α or Escherichia coli TOP10; the yeast is Pichia pastoris X33 or GS115.

[0024] The L-amino acid deaminase LAAD-ph3 is derived from... Proteus hauseri Its amino acid sequence is shown in SEQ ID NO: 1; the nucleotide sequence of its gene encoding L-amino acid deaminase LAAD-ph3 is shown in SEQ ID NO: 2.

[0025] A method for obtaining the above-mentioned mutant includes the following steps: expressing the gene encoding the L-amino acid deaminase LAAD-ph3 with the amino acid sequence shown in SEQ ID NO: 1 by site-directed saturation mutagenesis to obtain the L-amino acid deaminase mutant.

[0026] Furthermore, a mutation was introduced into the gene encoding the L-amino acid deaminase LAAD-ph3, as shown in SEQ ID NO: 1, using site-directed saturation mutagenesis. After the sequence was confirmed to be correct, the mutant was transformed into E. coli for expression, thus obtaining the L-amino acid deaminase mutant.

[0027] Preferably, the Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli DH5α, or Escherichia coli TOP10, etc.

[0028] A method for producing α-ketoglutaric acid includes the following steps: Recombinant microorganisms from the aforementioned mutant-related biological materials or recombinant microorganisms from L-amino acid deaminase LAAD-ph3-related biological materials were used as whole-cell catalysts to convert substrates containing L-glutamic acid to obtain α-ketoglutarate.

[0029] Furthermore, the reaction system containing 1.5–2.0 g / L whole cells, 10–15 mM L-glutamic acid, and 5–10 mM magnesium or calcium ions was reacted at 30–35 °C and pH 7.0–8.0 for 8–24 h.

[0030] Furthermore, a reaction system containing 1.5 g / L L-amino acid deaminase LAAD-ph3 whole cells, 10 mM L-glutamic acid, and 5 mM magnesium ions was reacted at 30℃ and pH 7.0 for 24 h; it was able to convert more than 0.6 mM of L-glutamic acid, with a molar yield of α-KG of more than 6%.

[0031] Furthermore, when a reaction system containing 1.5 g / L L-amino acid deaminase mutant LAAD-phRK whole cells, 10 mM L-glutamic acid, and 10 mM calcium ions was reacted at 30℃ and pH 8.0 for 8 h, it was able to convert more than 6 mM of L-glutamic acid, with a molar yield of α-KG of more than 60%.

[0032] The whole cells were collected as follows: the seed culture of recombinant microorganisms from the above-mentioned mutant-related biological materials or recombinant microorganisms from L-amino acid deaminase LAAD-ph3-related biological materials was transferred to the fermentation medium at an inoculation rate of 4-6% (more importantly, 5%), and fermented until OD. 600When the value is 0.8-1.4, add IPTG to a final concentration of 0.1 mM, induce culture, and collect cells.

[0033] Preferably, the seed culture medium is prepared as follows: a single colony of recombinant microorganisms from the above-mentioned mutant-related biological materials or recombinant microorganisms from L-amino acid deaminase LAAD-ph3-related biological materials is inoculated into LB liquid medium and cultured with shaking to obtain the seed culture medium.

[0034] The conditions for the shaking culture were 37±1℃ and 180±20 rpm for 10–12 h.

[0035] Preferably, the fermentation medium includes TB medium.

[0036] Preferably, the induction culture conditions are 16±1℃ and 180±20 rpm in a shaker for 16 to 20 h.

[0037] Preferably, the collected cells are collected by centrifugation, with centrifugation conditions of 2-8°C and 8000-10000 rpm for 10-15 min; more preferably, centrifugation at 4°C and 8000 rpm for 10 min.

[0038] The present invention has the following advantages and effects compared with the prior art: (1) Sequence analysis and comparison were performed on the amino acid sequence SEQ ID NO: 1 and its encoded nucleotide sequence SEQ ID NO: 2 determined by the present invention with other reported L-amino acid deaminases LAAD. The results showed that: Proteus hauseri The amino acid sequence of L-amino acid deaminase LAAD-ph3, SEQ ID NO: 1, is similar to that of Proteus mirabilis. Proteus mirabilis The similarity to the L-amino acid deaminase pm1 was 92.36%; and it was similar to that of Proteus vulgaris. Proteus vulgaris The similarity to L-amino acid deaminases was 83.07%; it was also similar to *Proteus mirabilis*. Proteus myxofaciens The similarity of the L-amino acid deaminases was 58.43%.

[0039] Among them, compared with pm1, which has the highest similarity, the differences are mainly reflected in: (1) 34 amino acids are significantly different at positions 4, 23, 48, 50, 74, 79, 149, 156-158, 169, 171, 172, 179, 217, 219, 222, 224, 236, 246, 251, 259, 304, 362, 366, 371, 372, 374, 393, 423, 449, 458, 460, and 463; (2) In the secondary... Structurally, due to the difference in amino acids 169-172, LAAD-ph3 has one more β-sheet than pm1 in the 169-174 amino acid region; in the 219-222 amino acid region, LAAD-ph3 exhibits an α-helix, while pm1 exhibits a β-sheet; in the 420-427 amino acid region, LAAD-ph3 has one more α-helix than pm1; and at the 447-450 amino acid region, LAAD-ph3 exhibits an α-helix, while pm1 exhibits a β-sheet.

[0040] It is evident that the L-amino acid deaminase LAAD-ph3 of this invention is a novel L-amino acid deaminase, which has not been previously reported in domestic or international literature.

[0041] (2) This invention yielded a total of 8 mutants, including 4 single-point mutation mutants and 4 combined mutants; compared with the wild type, the catalytic activity of the mutants in the production of α-ketoglutarate from L-glutamic acid was significantly improved. Among them, the mutants obtained through screening... Proteus hauseri The amino acid sequence of the L-amino acid deaminase mutant LAAD-phRK is shown in SEQ ID NO: 3. Compared with the wild type, the yield of α-ketoglutarate is increased by 4.53 times. Moreover, the amino acid sequence of this L-amino acid deaminase mutant LAAD-phRK has not been reported in domestic or foreign literature before.

[0042] (3) The present invention provides Proteus hauseri The L-amino acid deaminase mutant LAAD-phRK has the ability to catalyze the conversion of L-glutamic acid to α-ketoglutarate. 1.5 g / L LAAD-phRK whole-cell catalyst in a reaction system of 10 mM L-glutamic acid and 10 mM calcium ions, reacted at 30℃ and pH 8.0 for 8 h, can convert more than 6 mM of L-glutamic acid, with a molar yield of α-KG of more than 60%.

[0043] In summary, the present invention Proteus hauseri The L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK derived from this source can efficiently synthesize α-ketoglutarate, and have broad prospects for large-scale industrial applications. Attached Figure Description

[0044] Figure 1 This is an SDS-PAGE electrophoresis result of recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK in Example 1; where M: protein standard molecular weight marker (10-190kDa); lane 1: recombinant Escherichia coli BL21(DE3)-pET-28a-LAAD-ph3; lane 2: blank control; lane 3: recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-phRK.

[0045] Figure 2 This is an SDS-PAGE electrophoresis result of recombinant Escherichia coli DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK in Example 2; M: protein standard molecular weight marker (10-190kDa); Lane 1: blank control; Lane 2: recombinant Escherichia coli DH5α / pET-26b-LAAD-phRK; Lane 3: recombinant Escherichia coli DH5α / pET-26b-LAAD-ph3.

[0046] Figure 3 This is an SDS-PAGE electrophoresis result of recombinant Escherichia coli TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK in Example 3; M: protein standard molecular weight marker (10-190kDa); Lane 1: recombinant Escherichia coli TOP10 / pET-28a-LAAD-phRK; Lane 2: blank control; Lane 3: recombinant Escherichia coli TOP10 / pET-28a-LAAD-ph3.

[0047] Figure 4 This is a graph showing the determination of the molar production rate of α-KG catalyzed by the enzyme LAAD-ph3 mutant expressed in recombinant Escherichia coli in Example 5.

[0048] Figure 5 This is a comparison diagram of the amino acid sequences of L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0050] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0051] Example 1: Construction of L-amino acid deaminases LAAD-ph3 and LAAD-phRK encoding genes in Escherichia coli BL21(DE3) genetically engineered bacteria and preparation of whole-cell catalysts. Sangon Biotech Co., Ltd. was commissioned to synthesize the full-length gene encoding the corresponding amino acid sequences according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4), and then utilized... Nco I / Xho I The plasmids were digested with the corresponding enzymes and ligated into the same digested pET-28a(+) plasmid. Positive transformants were screened by sequencing to obtain positive recombinant plasmids pET-28a-LAAD-ph3 and pET-28a-LAAD-phRK. The constructed plasmids were then transformed into E. coli BL21(DE3) competent cells to construct engineered bacteria BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK.

[0052] Engineered bacteria BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK were inoculated into 10 mL of LB liquid medium (50 μg / mL kanamycin sulfate) and cultured at 37°C and 180 rpm for 10–12 h to obtain seed culture. The seed culture was then transferred at a 5% inoculation rate to TB medium (50 μg / mL kanamycin sulfate) and cultured at 37°C and 180 rpm. When the bacteria reached the OD value... 600 When the pH value was 0.8-1.4, IPTG was added to a final concentration of 0.1 mM, and the cells were induced and cultured in a shaker at 16 ℃ and 180 rpm for 20 h. After induction, the fermentation broth was centrifuged to stop fermentation, and the cells were collected. The centrifugation conditions were: 4 ℃, 8000 rpm, 10 min. After centrifugation, the cells were washed with 5 mL of PBS buffer, and centrifuged again. This step was repeated once. Finally, the cells were resuspended in 5 mL of PBS buffer and stored at 4 ℃ for later use, yielding the L-amino acid deaminase BL21(DE3)-pET-28a-LAAD-ph3 and the whole-cell catalyst BL21(DE3)-pET-28a-LAAD-phRK.

[0053] The whole-cell catalysts BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK were analyzed by gel electrophoresis using 12% (w / v) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE.

[0054] The conditions for its gel electrophoresis are as follows: Sample preparation: Take 10 μL of loading buffer (5x), mix 40 μL of sample with loading buffer (5x) at a ratio of 4:1, and boil (95℃) for 5 min; Sample loading volume: 10 μL; Marker loading volume: 5 μL; Voltage 150 mV, current 90 mA, duration 90 min.

[0055] 1x Electrode Buffer (pH 8.3): Glycine 14.4g, Tris 3g, SDS 1g, dissolved in distilled water and brought to a final volume of 1L; Coomassie Brilliant Blue R-250 staining solution (500 mL): Add 0.23 g Coomassie Brilliant Blue R250, 250 mL methanol, 50 mL acetic acid, and distilled water to a final volume of 500 mL; Decolorizing solution (1L): 100 mL methanol, 100 mL acetic acid, add distilled water to make up to 1 L.

[0056] Electrophoresis results as follows Figure 1 As shown, lane 1 is recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-ph3, and lane 3 is recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-phRK. Compared with the control group lane 2, a clear protein band (at the 52kDa position) appears in the protein expressed by the engineered bacteria.

[0057] Example 2: Construction of L-amino acid deaminases LAAD-ph3 and LAAD-phRK encoding genes in *Escherichia coli* DH5α genetically engineered bacteria and preparation of whole-cell catalysts. Sangon Biotech Co., Ltd. was commissioned to synthesize the full-length gene encoding the corresponding amino acid sequences according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4), and then utilized... Nco I / Xho I The plasmids were digested with the appropriate enzymes and ligated into the same digested pET-26b(+) plasmid. Positive transformants were screened by sequencing to obtain the positive recombinant plasmids pET-26b-LAAD-ph3 and pET-26b-LAAD-phRK. The constructed plasmids were then transformed into E. coli DH5α competent cells to construct the engineered bacteria DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK.

[0058] The whole-cell catalysts for L-amino acid deaminase DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK were prepared using the same method as in Example 1.

[0059] The whole-cell catalysts DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK were analyzed by gel electrophoresis using 12% (w / v) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE, the same method as in Example 1.

[0060] Electrophoresis results as follows Figure 2 As shown, lane 3 is recombinant Escherichia coli DH5α / pET-26b-LAAD-ph3, and lane 2 is recombinant Escherichia coli DH5α / pET-26b-LAAD-phRK. Compared with the control group lane 1, a clear protein band (at the 52kDa position) appears in the protein expressed by the engineered bacteria.

[0061] Example 3: Construction of L-amino acid deaminases LAAD-ph3 and LAAD-phRK encoding genes in Escherichia coli TOP10 genetically engineered bacteria and preparation of whole-cell catalysts. Sangon Biotech Co., Ltd. was commissioned to synthesize the full-length gene encoding the corresponding amino acid sequences according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4), and then utilized... Nde I / Xho I The plasmids were digested with the corresponding enzymes and ligated into the same digested pET-28a(+) plasmid. Positive transformants were screened by sequencing to obtain the positive recombinant plasmids pET-28a-LAAD-ph3 and pET-28a-LAAD-phRK. The constructed plasmids were then transformed into E. coli TOP10 competent cells to construct the engineered bacteria TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK.

[0062] The whole-cell catalysts for L-amino acid deaminase TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK were prepared using the same method as in Example 1.

[0063] The TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK whole-cell catalysts were analyzed by gel electrophoresis using 12% (w / v) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE, the same method as in Example 1.

[0064] Electrophoresis results as follows Figure 3As shown, lane 3 is recombinant E. coli TOP10 / pET-28a-LAAD-ph3, and lane 1 is recombinant E. coli TOP10 / pET-28a-LAAD-phRK. Compared with the control group lane 2, a clear protein band (at the 52kDa position) appeared in the expressed protein of the engineered bacteria.

[0065] Example 4: Recombinant expression of L-amino acid deaminase LAAD-ph3 catalyzing the synthesis of α-ketoglutarate The prepared 1.5 g / L recombinant expression BL21(DE3) / pET-28a-LAAD-ph3 whole-cell catalyst was placed in a reaction system of 10 mM L-glutamic acid and 5 mM magnesium ions and reacted with shaking for 24 h at 30 °C and pH 7.0.

[0066] The molar production rate of α-KG catalyzed by the whole-cell catalyst BL21(DE3) / pET-28a-LAAD-ph3 was determined.

[0067] The method for determining the molar formation rate of α-KG is as follows: The reaction system consisted of 10 mM L-glutamic acid (L-Glu), 5 mM magnesium ions, and 1.5 g / L whole-cell catalyst. The reaction was carried out at 30℃ and pH 7.0 with shaking for 24 h. The reaction was terminated by centrifugation for 5 min to remove bacterial cells. The supernatant was collected, and the α-KG content was determined by a colorimetric method.

[0068] The colorimetric method for determining α-KG content is as follows: Take 50 μL of supernatant and mix it with 100 μL of 0.1% 2,4-dinitrophenylhydrazine. After standing at room temperature for 5 min, add 1 mL of 1.5 M sodium hydroxide solution. After the color stabilizes, pipette 200 μL of the liquid into a 96-well plate and place it in a microplate reader to detect its absorbance at a wavelength of 520 nm.

[0069] The method for establishing the α-KG standard curve is as follows: Six sets of α-KG standard solutions (0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM) were prepared using α-KG standards. The absorbance was recorded using an enzyme-linked immunosorbent assay (ELISA) reader. A linear regression was performed on the experimental data using data processing software, with the α-KG concentration on the x-axis and the absorbance measured by the ELISA reader on the y-axis, to obtain the α-KG standard curve.

[0070] The formula for calculating molar generation rate is as follows:

[0071] The results showed that BL21(DE3) / pET-28a-LAAD-ph3 could convert more than 0.6 mM of L-glutamic acid by reacting at 30 ℃ and pH 7.0 for 24 h, with a molar yield of 6.24% for α-KG.

[0072] Similarly, following the method of this embodiment, DH5α / pET-26b-LAAD-ph3 reacted at 30℃ and pH 7.0 for 24 h to convert more than 0.6 mM of L-glutamic acid, with a molar yield of α-KG of 6.07%. TOP10 / pET-28a-LAAD-ph3 reacted at 30℃ and pH 7.0 for 24 h to convert more than 0.6 mM of L-glutamic acid, with a molar yield of α-KG of 6.60%.

[0073] Example 5 Construction of the L-amino acid deaminase mutant LAAD-phRK The three-dimensional structure of LAAD-ph3 was predicted using Alphafold3, and molecular docking between the protein model LAAD-ph3 and L-glutamate was performed using the automated docking software AutoDdck 4.0. By analyzing key sites in the model's active site and the interactions between the substrate L-glutamate and FAD with the enzyme, site-directed saturation mutagenesis was performed at four amino acid sites: ILE64 (I64), GLY257 (G257), GLN278 (Q278), and MET440 (M440). Using the amino acid sequence SEQ ID NO: 1 of the L-amino acid deaminase LAAD-ph3 as a template, these four sites were mutated, and the corresponding coding genes were synthesized and transformed into *E. coli* BL21(DE3) to construct a whole-cell catalyst from the mutant engineered bacteria. This catalyst was then used to catalyze the synthesis of α-KG, and its yield was measured.

[0074] The following conditions were used to construct whole-cell catalysts and catalytic synthesis reactions: Pick up the bacteria from the glycerol tube and streak them onto solid LB medium (50 μg / mL kanamycin sulfate), then incubate overnight at 37 ℃. The next day, pick a single colony and inoculate it into 10 mL of LB liquid medium (50 μg / mL kanamycin sulfate), and incubate at 37 ℃ and 180 rpm for 10–12 h to obtain the seed culture. Transfer the seed culture to TB medium (50 μg / mL kanamycin sulfate) at a 5% inoculation rate and incubate at 37 ℃ and 180 rpm. When the bacteria grow to the OD value… 600When the pH value was 1.2, IPTG was added to a final concentration of 0.1 mM, and the cells were induced and cultured in a shaker at 16℃ and 180 rpm for 20 h. After induction, the fermentation broth was centrifuged to stop fermentation, and the cells were collected. The centrifugation conditions were: 4℃, 8000 rpm, 10 min. After centrifugation, the cells were washed with 5 mL of PBS buffer, centrifuged again, and this step was repeated once. Finally, the cells were resuspended in 5 mL of PBS buffer and stored at 4℃ for later use, thus obtaining the L-amino acid deaminase mutant whole-cell catalyst.

[0075] The L-amino acid deaminase mutant whole-cell catalyst was used for the synthesis of α-KG at 30°C and pH 7.0. The reaction system consisted of 10 mM L-glutamic acid, 5 mM magnesium ions, and 1.5 g / L mutant whole-cell catalyst. The reaction was terminated by shaking at 180 rpm for 24 h, centrifuging for 5 min to remove bacterial cells, and collecting the supernatant. The α-KG content was determined by a colorimetric method under the same conditions as in Example 4.

[0076] The measurement results are as follows Figure 4 As shown, the results indicate that the catalytic activity of mutants obtained through various mutation methods at these four mutation sites was significantly enhanced. Among them, the LAAD-phRK mutant (I64R / G257H / Q278R / M440K) exhibited the highest α-KG yield after recombinant expression, at 34.53%, which was 4.53 times higher than the control group. This was followed by I64R / Q278R / M440K, I64R / M440K, Q278R, Q278R / M440K, M440K, I64R, and G257H, with yields of 27.63%, 26.13%, 26.02%, 17.81%, 17.73%, 16.91%, and 10.08%, respectively, representing increases of 3.43 times, 3.19 times, 3.17 times, 1.85 times, 1.84 times, 1.71 times, and 0.61 times compared to the control group.

[0077] The mutant LAAD-phRK was verified by sequencing, and its mutated amino acids are as follows: ILE (I) at site 64 is mutated to ARG (R), GLY (G) at site 257 is mutated to HIS (H), GLN (Q) at site 278 is mutated to ARG (R), and MET (M) at site 440 is mutated to LYS (K).

[0078] Among them, I64R: the codon is mutated from ATT to CGT; G257H: the codon is mutated from GGT to CAT; Q278R: the codon is mutated from CAG to CGT; M440K: the codon is mutated from ATG to AAG.

[0079] The amino acid sequence of the L-amino acid deaminase mutant LAAD-phRK, obtained through screening, is shown in SEQ ID NO: 3, and the optimized nucleotide sequence is shown in SEQ ID NO: 4. A comparison diagram of the amino acid sequences of the L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK is shown below. Figure 5 As shown.

[0080] Similarly, referring to the method of this embodiment, the molar yield of α-KG synthesized by mutant whole-cell catalyst obtained with Escherichia coli DH5α or TOP10 as host is comparable to the molar yield of α-KG synthesized by mutant whole-cell catalyst obtained with Escherichia coli BL21(DE3) as host.

[0081] Example 6: Synthesis of α-ketoglutaric acid catalyzed by the mutant LAAD-phRK The recombinant E. coli strain BL21(DE3) expressing the L-amino acid deaminase mutant LAAD-phRK was used to prepare whole-cell catalysts under optimized conditions: The catalysts were picked from glycerol tubes and streaked onto solid LB medium (50 μg / mL kanamycin sulfate) and incubated overnight at 37 °C. The next day, single colonies were picked and inoculated into 10 mL of LB liquid medium (50 μg / mL kanamycin sulfate) and cultured at 37 °C and 180 rpm for 10–12 h to obtain seed culture. The seed culture was then transferred at a 5% inoculation rate to TB medium (50 μg / mL kanamycin sulfate) and cultured at 37 °C and 180 rpm. When the bacteria reached the OD value... 600 When the pH value was 1.0, IPTG was added to a final concentration of 0.1 mM, and the cells were induced and cultured in a shaker at 16℃ and 180 rpm for 16 h. After induction, the fermentation broth was centrifuged to stop fermentation, and the cells were collected. The centrifugation conditions were: 4℃, 8000 rpm, 10 min. After centrifugation, the cells were washed with 5 mL of PBS buffer, and centrifuged again. This step was repeated once. Finally, the cells were resuspended in 5 mL of PBS buffer and stored at 4℃ for later use, yielding the L-amino acid deaminase mutant LAAD-phRK whole-cell catalyst.

[0082] Optimized catalytic synthesis reaction conditions: The L-amino acid deaminase mutant LAAD-phRK whole-cell catalyst was used for the synthesis of α-KG at 30℃ and pH 8.0. The reaction system consisted of 10 mM L-glutamic acid, 10 mM calcium ions, and 1.5 g / L of the mutant LAAD-phRK whole-cell catalyst. The reaction was terminated by shaking at 180 rpm for 8 h, centrifuging at high speed for 5 min to remove bacterial cells, collecting the supernatant, and detecting the α-KG content using a colorimetric method under the same conditions as in Example 4. This recombinant Escherichia coli mutant strain LAAD-phRK, reacting at 30℃ and pH 8.0 for 8 h, could convert more than 6 mM of L-glutamic acid, with a molar yield of 64.89% for α-KG.

[0083] Similarly, referring to the method of this embodiment, DH5α / pET-26b-LAAD-phRK reacted at 30°C and pH 8.0 for 8 hours to convert more than 6 mM of L-glutamic acid, with a molar yield of α-KG of 60.84%. TOP10 / pET-28a-LAAD-phRK reacted at 30°C and pH 8.0 for 8 hours to convert more than 6 mM of L-glutamic acid, with a molar yield of α-KG of 68.12%.

[0084] In summary, the L-amino acid deaminase LAAD of the present invention can stably and efficiently convert L-glutamic acid into α-ketoglutarate.

[0085] The above experimental results further demonstrate that the present invention can be safely and efficiently used in actual production, thereby effectively reducing actual cost input.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An L-amino acid deaminase mutant, characterized by: The amino acid sequence of the mutant is obtained by any one of the following mutations of SEQ ID NO: 1: I64R, G257H, M440K, or I64R / M440K.

2. The gene encoding the L-amino acid deaminase mutant of claim 1.

3. The biomaterial related to the L-amino acid deaminase mutant as described in claim 1, characterized in that: It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2; (b) A recombinant expression vector containing the gene of claim 2; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant microorganism containing the gene of claim 2; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

4. The biomaterial according to claim 3, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is a pET series vector or a pPICZα vector; The host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from prokaryotes or yeast.

5. The use of the gene of claim 2 or the biomaterial of any one of claims 3 to 4 in the preparation of L-amino acid deaminase mutants.

6. The application of the L-amino acid deaminase mutant of claim 1, the gene of claim 2, or the biomaterial of any one of claims 3 to 4 in the biosynthesis of α-ketoglutarate.

7. A method for obtaining the L-amino acid deaminase mutant of claim 1, characterized in that, The method includes the following steps: expressing the gene encoding the L-amino acid deaminase LAAD-ph3, as shown in SEQ ID NO: 1, through site-directed saturation mutagenesis to obtain the L-amino acid deaminase mutant of claim 1.

8. A method for producing α-ketoglutaric acid, characterized in that, Includes the following steps: Using the recombinant microorganism described in any one of claims 3 to 4 as a whole-cell catalyst, α-ketoglutarate is obtained by converting a substrate containing L-glutamic acid.

9. The method according to claim 8, characterized in that: The reaction system containing 1.5–2.0 g / L whole cells, 10–15 mM ML-glutamate, and 5–10 mM magnesium or calcium ions was reacted at 30–35 °C and pH 7.0–8.0 for 8–24 h.

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