An L-amino acid deaminase, its mutants and applications

Abstract

The present invention discloses an L-amino acid deaminase and its mutants and applications. The present invention for the first time verifies that the L-amino acid deaminase derived from Proteus hauseri has the ability to catalyze the formation of α-KG from L-glutamic acid. Through protein three-dimensional structure simulation, etc., the related sites that may affect catalysis and binding are predicted to be I64, G257, Q278, and M440. Site-directed saturation mutagenesis is carried out to obtain mutants and engineering bacteria with high-efficiency catalytic synthesis of α-KG. Compared with the wild type, the yield of α-KG catalyzed by the mutant LAAD-phRK is increased by 4.53 times. 1.5 g / L of LAAD-phRK whole cells in a reaction system of 10 mM L-glutamic acid and 10 mM calcium ions can convert more than 6 mM of L-glutamic acid at 30 °C and pH 8.0 for 8 h, and the molar yield of α-KG is above 60%, showing broad prospects for large-scale industrial applications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to an Proteus hauseri L-amino acid deaminase and its mutants derived therefrom and applications. Background Art

[0002] α-Ketoglutarate (α-KG) is one of the important intermediate products of the tricarboxylic acid cycle and is involved in the synthesis of amino acids, vitamins, and organic acids and energy metabolism in organisms. α-Ketoglutarate forms L-glutamate through transamination and enters nitrogen metabolism, which is a key node connecting carbon metabolism and nitrogen metabolism in cells. Common α-ketoglutarate derivatives include γ-aminobutyric acid (GABA), 5-aminolevulinic acid (5-ALA), and hydroxides such as 4-hydroxyisoleucine (4-HIL) catalyzed by α-ketoglutarate-dependent hydroxylases. So far, α-ketoglutarate and its derivatives have been widely used in many fields such as chemical industry, food, medicine, and daily chemicals, and have very broad application prospects.

[0003] Currently, the synthesis methods of α-ketoglutarate mainly include chemical methods, microbial fermentation methods, and enzyme-catalyzed methods. In the chemical production of α-ketoglutarate, diethyl succinate, diethyl oxalate, etc. are usually used as raw materials, and α-ketoglutarate is synthesized through processes such as condensation, hydrolysis, distillation concentration, and crystallization, with a maximum yield of 75%. However, the starting materials for the chemical production of α-KG have special structures, contain toxic substances that pollute the environment, require multiple steps of reaction, have a low final yield, and have serious safety problems, which limit their application in industries such as food and cosmetics. For decades, the production of α-KG by microbial fermentation has been continuously developed and made great progress, but there are also disadvantages such as a lack of production strain resources, low yields of metabolites, difficult separation, and long production cycles, which cannot meet the needs of large-scale industrial production. The enzyme-catalyzed conversion method has the advantages of mild reaction conditions, single product, simple extraction process, high product concentration, and high purification yield. Therefore, the enzyme-catalyzed production of α-ketoglutarate has become the mainstream at present. There are mainly three paths for the enzyme-catalyzed synthesis of α-ketoglutarate, all using L-glutamate as the raw material, and respectively involving glutamate dehydrogenase (GDH), L-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.

[0004] Since the affinity of glutamate dehydrogenase for α-ketoglutaric acid is higher than that for glutamate and this reaction requires the coenzyme nicotinamide adenine dinucleotide phosphate (NADP + ), the yield of α-ketoglutaric acid is extremely low. L-amino acid oxidase in Path 2 produces hydrogen peroxide during the reaction, which severely inhibits the enzyme activity. Although adding catalase (CAT) can eliminate hydrogen peroxide, it is difficult to be industrially applied due to the complexity of the production process and the increase in production costs. L-amino acid deaminase in Path 3 also catalyzes the synthesis of α-ketoglutaric acid from L-glutamate as a substrate. Only α-ketoglutaric acid, ammonia, and water are produced during the reaction, and no toxic substance such as H2O2 is generated, which has little impact on cell growth. Comparatively, it is more conducive to the production of α-ketoglutaric acid. Currently, it is known from research that L-amino acid deaminase (pm1) derived from Proteus mirabilis ( Proteus mirabilis ) can be heterologously expressed in Escherichia coli BL21(DE3), effectively converting L-glutamate into α-ketoglutaric acid. However, as a membrane protein, the expression level of pm1 is low, and its specificity for the substrate L-glutamate is not high (35.8%). Currently, there is little research on L-amino acid deaminase, and very few types of L-amino acid deaminases that can effectively catalyze the synthesis of α-ketoglutaric acid have been discovered. Only pm1 from Proteus mirabilis has been reported, which limits the application of the enzyme-catalyzed method for synthesizing α-ketoglutaric acid in industry. Therefore, exploring new L-amino acid deaminases with high efficiency and improving their enzyme activity through directed evolution will help further reduce the cost of industrial production. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the existing α-ketoglutaric acid synthesis process, the primary object of the present invention is to provide an Proteus hauseri L-amino acid deaminase and its mutants derived from

[0006] Another object of the present invention is to provide the coding genes of the above L-amino acid deaminase and its mutants.

[0007] Another object of the present invention is to provide the application of the above L-amino acid deaminase and its mutants in the biosynthesis of α-ketoglutaric acid.

[0008] The present invention first verified Proteus hauseri ( Proteus hauseriThe L-amino acid deaminase from the source has the ability to catalyze the formation of α-ketoglutaric acid from L-glutamic acid. Further, through protein three-dimensional structure simulation, molecular docking, and virtual saturation mutagenesis, the related 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 with high-efficiency catalytic synthesis of α-ketoglutaric acid, which have good application prospects.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] An L-amino acid deaminase mutant, whose amino acid sequence is obtained by any one of the following mutations of SEQ ID NO: 1:

[0011] At least one of I64R, G257H, Q278R, and M440K; wherein, I64R means that the 64th amino acid is mutated from ILE (I) to ARG (R), and the same applies to others.

[0012] Furthermore, the L-amino acid deaminase mutant, whose amino acid sequence is obtained by any one of the following mutations of SEQ ID NO: 1:

[0013] I64R, G257H, Q278R, M440K, I64R / M440K, Q278R / M440K, I64R / Q278R / M440K, or I64R / G257H / Q278R / M440K.

[0014] Preferably, the gene sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2.

[0015] A coding gene for the above L-amino acid deaminase mutant.

[0016] Preferably, an L-amino acid deaminase mutant LAAD-phRK (I64R / G257H / Q278R / M440K), whose amino acid sequence is as shown in SEQ ID NO: 3.

[0017] A coding gene for the above L-amino acid deaminase mutant LAAD-phRK, whose nucleotide sequence is as shown in SEQ ID NO: 4.

[0018] The biological material related to the above mutant is any one or a combination of the following biological materials:

[0019] (a) An expression cassette containing the above coding gene;

[0020] (b) A recombinant expression vector containing the above coding gene;

[0021] (c) A recombinant expression vector containing the expression cassette described in (a);

[0022] (d) A recombinant microorganism containing the above-mentioned coding gene;

[0023] (e) A recombinant microorganism containing the expression cassette described in (a);

[0024] (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

[0025] Further, the starting vectors of the recombinant expression vectors described in (b) and (c) are vectors such as pET series vectors or pPICZα vectors, etc.; preferably pET-28a(+) vector, pET-26b(+) or pPICZαA vector.

[0026] Further, the host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from prokaryotes or yeasts, etc.; the prokaryotes include bacteria such as Escherichia genus ( Escherichia ); the yeasts include yeasts such as Pichia pastoris. More specifically, the prokaryote is Escherichia coli ( Escherichia coli , E. coli ), specifically Escherichia coli BL21(DE3), Escherichia coli DH5α or Escherichia coli TOP10; the yeast is Pichia pastoris X33 or GS115.

[0027] Use of the above-mentioned mutant, coding gene, and biological materials related to the mutant in the preparation of L-amino acid deaminase mutant.

[0028] Further, the uses of the mutant, coding gene, biological materials related to the mutant, L-amino acid deaminase LAAD-ph3, and biological materials related to L-amino acid deaminase LAAD-ph3 are one of the following uses:

[0029] (I) Use in the biosynthesis of α-ketoglutaric acid;

[0030] (II) Use in food, pharmaceuticals, feeds, cosmetics, and chemicals;

[0031] (III) Use in health products.

[0032] The biological materials related to L-amino acid deaminase LAAD-ph3 are any one or a combination of the following biological materials:

[0033] 1) An expression cassette containing the gene encoding L-amino acid deaminase LAAD-ph3;

[0034] 2) A recombinant expression vector containing the gene encoding L-amino acid deaminase LAAD-ph3;

[0035] 3) A recombinant expression vector containing the expression cassette described in 1);

[0036] 4) A recombinant microorganism containing the gene encoding L - amino acid deaminase LAAD - ph3;

[0037] 5) A recombinant microorganism containing the expression cassette described in 1);

[0038] 6) A recombinant microorganism containing the recombinant expression vector described in 2) or 3).

[0039] Furthermore, the starting vector of the recombinant expression vectors described in 2) and 3) is a vector of the pET series or the pPICZα vector, etc.; preferably, it is the pET - 28a(+) vector, pET - 26b(+) or the pPICZαA vector.

[0040] Furthermore, the host microorganism corresponding to the recombinant microorganisms described in 4), 5), and 6) is selected from prokaryotes or yeasts, etc.; the prokaryotes include bacteria such as Escherichia Escherichia ), etc.; the yeasts include yeasts such as Pichia pastoris. 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.

[0041] Among them, the L - amino acid deaminase LAAD - ph3 is derived from Proteus hauseri , and its amino acid sequence is as shown in SEQ ID NO: 1; the nucleotide sequence of the gene encoding L - amino acid deaminase LAAD - ph3 is as shown in SEQ ID NO: 2.

[0042] A method for obtaining the above - mentioned mutant includes the following steps: performing site - directed mutagenesis on the gene encoding L - amino acid deaminase LAAD - ph3 with an amino acid sequence as shown in SEQ ID NO: 1 by site - directed saturation mutagenesis technology and then expressing it to obtain an L - amino acid deaminase mutant.

[0043] Furthermore, introducing mutations into the gene encoding L - amino acid deaminase LAAD - ph3 with an amino acid sequence as shown in SEQ ID NO: 1 by site - directed saturation mutagenesis technology, after correct sequencing, transforming it into Escherichia coli for expression to obtain an L - amino acid deaminase mutant.

[0044] Preferably, the Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli DH5α or Escherichia coli TOP10, etc.

[0045] A method for producing α-ketoglutaric acid, comprising the following steps:

[0046] Using the recombinant microorganism in the biological material related to the above mutant or the recombinant microorganism in the biological material related to L-amino acid deaminase LAAD-ph3 as a whole-cell catalyst to convert the substrate containing L-glutamic acid to obtain α-ketoglutaric acid.

[0047] Furthermore, reacting a reaction system containing 1.5 - 2.0 g / L whole cells, 10 - 15 mM L-glutamic acid, and 5 - 10 mM magnesium ions or calcium ions at 30 - 35 °C and pH 7.0 - 8.0 for 8 - 24 h.

[0048] Even further, reacting a reaction system containing 1.5 g / L whole cells of L-amino acid deaminase LAAD-ph3, 10 mM L-glutamic acid, and 5 mM magnesium ions at 30 °C and pH 7.0 for 24 h; it can convert more than 0.6 mM of L-glutamic acid, and the molar yield of α-KG is above 6%.

[0049] Even further, reacting a reaction system containing 1.5 g / L whole cells of the L-amino acid deaminase mutant LAAD-phRK, 10 mM L-glutamic acid, and 10 mM calcium ions at 30 °C and pH 8.0 for 8 h; it can convert more than 6 mM of L-glutamic acid, and the molar yield of α-KG is above 60%.

[0050] The whole cells are collected as follows: Transfer the seed culture solution of the recombinant microorganism in the biological material related to the above mutant or the recombinant microorganism in the biological material related to L-amino acid deaminase LAAD-ph3 to the fermentation medium at an inoculation amount of 4 - 6% (further 5%), and ferment and culture until the OD 600 value is 0.8 - 1.4, add IPTG with a final concentration of 0.1 mM, induce culture, and collect the cells.

[0051] Preferably, the seed culture solution is prepared as follows: Inoculate a single colony of the recombinant microorganism in the biological material related to the above mutant or the recombinant microorganism in the biological material related to L-amino acid deaminase LAAD-ph3 into the LB liquid medium, and shake culture to obtain the seed culture solution.

[0052] The conditions for the shake culture are 37 ± 1 °C and 180 ± 20 rpm for 10 - 12 h.

[0053] Preferably, the fermentation medium includes TB medium.

[0054] Preferably, the induction culture is carried out at 16±1°C and 180±20 rpm in a shaking incubator for 16 to 20 h.

[0055] Preferably, the cells are collected by centrifugation, and the centrifugation conditions are 2-8°C, 8000-10000 rpm for 10-15 min; further, 4°C, 8000 rpm for 10 min.

[0056] Compared with the prior art, the present invention has the following advantages and effects:

[0057] (1) The amino acid sequence SEQ ID NO: 1 determined by the present invention and the nucleotide sequence SEQ ID NO: 2 encoded by the present invention were subjected to sequence analysis and comparison with other reported L-amino acid deaminase LAADs. The results showed that: Proteus hauseri The amino acid sequence of L-amino acid deaminase LAAD-ph3 of SEQ ID NO: 1 and Proteus mirabilis Proteus mirabilis The similarity of L-amino acid deaminase pm1 to Proteus vulgaris is 92.36%; Proteus vulgaris The similarity of L-amino acid deaminase to Proteus viscosus is 83.07%; Proteus myxofaciens The similarity with L-amino acid deaminases is 58.43%.

[0058] Among them, compared with pm1, which has the highest similarity, the differences are mainly reflected in: (1) there are 34 significant differences in amino acids 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) at 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 part; in the 219-222 amino acid part, LAAD-ph3 exhibits an α-helix, while pm1 exhibits a β-sheet; in the 420-427 amino acid part, LAAD-ph3 has one more α-helix than pm1; at 447-450 amino acids, LAAD-ph3 exhibits an α-helix, while pm1 exhibits a β-sheet.

[0059] It can be seen that the L-amino acid deaminase LAAD-ph3 of the present invention is a brand-new L-amino acid deaminase, which has not been reported in domestic and foreign literature before.

[0060] (2) A total of 8 mutants were obtained in this invention, including 4 single-point mutant mutants and 4 combined mutant mutants; compared with the wild type, the catalytic activities of the mutants in catalyzing L-glutamic acid to produce α-ketoglutaric acid were all significantly improved. Among them, the amino acid sequence of the L-amino acid deaminase mutant LAAD-phRK derived from Proteus hauseri is shown in SEQ ID NO: 3. Compared with the wild type, the yield of α-ketoglutaric acid produced by catalysis increased by 4.53 times, and the amino acid sequence of this L-amino acid deaminase mutant LAAD-phRK has not been reported in domestic and foreign literatures before.

[0061] (3) The L-amino acid deaminase mutant LAAD-phRK provided by this invention, derived from Proteus hauseri has the ability to efficiently catalyze L-glutamic acid to produce α-ketoglutaric acid. 1.5 g / L of LAAD-phRK whole-cell catalyst in a reaction system of 10 mM L-glutamic acid and 10 mM calcium ions, reacts at 30 °C and pH 8.0 for 8 h, and can convert more than 6 mM of L-glutamic acid, and the molar yield of α-KG is above 60%.

[0062] In summary, the L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK provided by this invention, derived from Proteus hauseri can efficiently synthesize α-ketoglutaric acid and have broad prospects for large-scale industrial applications. Description of the Drawings

[0063] Figure 1 is the SDS-PAGE electrophoresis result diagram of recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK in Example 1; among them, M: protein standard molecular weight marker (10 - 190 kDa); 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.

[0064] Figure 2 is the SDS-PAGE electrophoresis result diagram 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 - 190 kDa); lane 1: blank control; lane 2: recombinant Escherichia coli DH5α / pET-26b-LAAD-phRK, lane 3: recombinant Escherichia coli DH5α / pET-26b-LAAD-ph3.

[0065] Figure 3 It is the SDS-PAGE electrophoresis result diagram 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 - 190 kDa); Lane 1: Recombinant Escherichia coli TOP10 / pET-28a-LAAD-phRK; Lane 2: Blank control; Lane 3: Recombinant Escherichia coli TOP10 / pET-28a-LAAD-ph3.

[0066] Figure 4 It is the determination diagram of the molar production rate of α-KG catalyzed by the LAAD-ph3 mutant enzyme expressed by recombinant Escherichia coli in Example 5.

[0067] Figure 5 It is the amino acid sequence comparison diagram of L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK. Specific implementation mode

[0068] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation modes of the present invention are not limited thereto.

[0069] If the specific test conditions are not specified in the following implementation schemes, they are usually in accordance with the conventional test conditions or the test conditions recommended by the reagent company. The materials, reagents, etc. used, unless otherwise specified, are all reagents and materials obtained from commercial channels.

[0070] Example 1 Construction of genetic engineering bacteria of L-amino acid deaminase LAAD-ph3 and LAAD-phRK encoding genes transferred into Escherichia coli BL21(DE3) and preparation of whole cell catalysts

[0071] Entrust Sangon Biotech Co., Ltd. to synthesize the full-length genes 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 use Nco I / Xho I Perform corresponding enzyme digestion and ligate it to the pET-28a(+) plasmid digested with the same enzyme. Screen positive transformants through sequencing verification to obtain positive recombinant plasmids pET-28a-LAAD-ph3 and pET-28a-LAAD-phRK. Transfer the constructed plasmids into Escherichia coli BL21(DE3) competent cells to construct engineering bacteria BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK.

[0072] The engineered strains 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 in a shaker at 37°C and 180 rpm for 10 - 12 h to obtain seed culture solutions. The seed culture solutions were transferred to TB medium (50 μg / mL kanamycin sulfate) at an inoculation amount of 5%, and cultured in a shaker at 37°C and 180 rpm. When the OD 600 value reached 0.8 - 1.4, IPTG with a final concentration of 0.1 mM was added, and induced culture was carried out in a shaker at 16°C and 180 rpm for 20 h. The fermentation broth after the induced culture was centrifuged to terminate fermentation, and the cells were collected. The centrifugation conditions were: 4°C, 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 suspended in 5 mL of PBS buffer and stored at 4°C for standby, thus obtaining the whole-cell catalysts of L-amino acid deaminase BL21(DE3)-pET-28a-LAAD-ph3 and BL21(DE3)-pET-28a-LAAD-phRK.

[0073] The whole-cell catalysts of BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK were analyzed by 12% (W / V) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE electrophoresis.

[0074] The conditions for the gel electrophoresis were as follows:

[0075] Sample treatment: Take 10 μL of loading buffer (5x), mix 40 μL of the sample with the loading buffer (5x) at a ratio of 4:1, and boil (95°C) for 5 min;

[0076] Sample loading volume: 10 μL, Maker loading volume: 5 μL;

[0077] Voltage 150 mV, current 90 mA, duration 90 min.

[0078] 1x electrode buffer (pH8.3): 14.4 g of glycine, 3 g of Tris, 1 g of SDS, dissolved in distilled water and made up to 1 L;

[0079] Coomassie Brilliant Blue R-250 staining solution (500 mL): Add 0.23 g of Coomassie Brilliant Blue R250, 250 mL of methanol, 50 mL of acetic acid, and make up to 500 mL with distilled water;

[0080] Decolorization solution (1 L): 100 mL of methanol, 100 mL of acetic acid, and add distilled water to make up to 1 L.

[0081] The electrophoresis results are as Figure 1 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 lane 2, an obvious protein band (at the position of 52 kDa) appears in the expressed proteins of the engineered bacteria.

[0082] Example 2 Construction of genetically engineered bacteria of Escherichia coli DH5α into which the L-amino acid deaminase LAAD-ph3 and LAAD-phRK encoding genes are transferred and preparation of whole-cell catalysts

[0083] Entrust Sangon Biotech Co., Ltd. to synthesize the full-length genes 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 use Nco I / Xho I to perform corresponding enzyme digestion, and ligate them to the pET-26b(+) plasmid digested with the same enzyme. Positive transformants are screened by sequencing to obtain the positive recombinant plasmids pET-26b-LAAD-ph3 and pET-26b-LAAD-phRK. The constructed plasmids are transferred into Escherichia coli DH5α competent cells to construct the engineered bacteria DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK.

[0084] Prepare the whole-cell catalysts of L-amino acid deaminase DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK by the same method as in Example 1.

[0085] Perform gel electrophoresis analysis of the whole-cell catalysts of DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK using 12% (W / V) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE by the same method as in Example 1.

[0086] The electrophoresis results are as Figure 2 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 lane 1, an obvious protein band (at the position of 52 kDa) appears in the expressed proteins of the engineered bacteria.

[0087] Example 3: Construction of Genetically Engineered Escherichia coli TOP10 Strains with Genes Encoding L-Amino Acid Deaminase LAAD-ph3 and LAAD-phRK and Preparation of Whole-Cell Catalysts

[0088] Shanghai Sangon Biotech Co., Ltd. was commissioned to synthesize the full-length genes 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). Using Nde I / Xho I corresponding restriction enzyme digestion was performed and ligated to the pET-28a(+) plasmid digested with the same enzyme. Positive transformants were screened by sequencing verification to obtain the positive recombinant plasmids pET-28a-LAAD-ph3 and pET-28a-LAAD-phRK. The constructed plasmids were transferred into Escherichia coli TOP10 competent cells to construct the engineered strains TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK.

[0089] The whole-cell catalysts of L-amino acid deaminase TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK were prepared by the same method as in Example 1.

[0090] The whole-cell catalysts of TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK were analyzed by 12% (W / V) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by the same method as in Example 1.

[0091] The electrophoresis results are as Figure 3 shown. Lane 3 is recombinant Escherichia coli TOP10 / pET-28a-LAAD-ph3, and lane 1 is recombinant Escherichia coli TOP10 / pET-28a-LAAD-phRK. Compared with lane 2 of the control group, an obvious protein band (at the position of 52 kDa) appeared in the expressed proteins of the engineered strains.

[0092] Example 4: Catalytic Synthesis of α-Ketoglutaric Acid by Recombinant Expression of L-Amino Acid Deaminase LAAD-ph3

[0093] 1.5 g / L of the prepared whole-cell catalyst of recombinant expression BL21(DE3) / pET-28a-LAAD-ph3 was added to a reaction system containing 10 mM L-glutamic acid and 5 mM magnesium ions, and the reaction was carried out with shaking at 30 °C and pH 7.0 for 24 h.

[0094] The molar formation rate of α-KG catalyzed by the whole-cell catalyst BL21(DE3) / pET-28a-LAAD-ph3 for the synthesis of L-Glu was determined.

[0095] The method for determining the molar formation rate of α-KG is as follows:

[0096] Reaction system: 10 mM L-glutamic acid (L-Glu), 5 mM magnesium ions and 1.5 g / L whole-cell catalyst. Under the conditions of 30 °C and pH 7.0, the reaction was oscillated for 24 h, and the reaction was terminated by high-speed centrifugation for 5 min to remove the cells. The supernatant was collected, and the α-KG content was detected by the colorimetric method.

[0097] The method for detecting the α-KG content by the colorimetric method is as follows:

[0098] Take 50 μL of the 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 is stable, pipette 200 μL of the liquid into a 96-well plate and detect its absorbance at a wavelength of 520 nm in an enzyme-linked immunosorbent assay reader.

[0099] The method for establishing the α-KG standard curve is as follows:

[0100] Six groups of α-KG standard solutions with concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM were prepared using α-KG standard products. Detection was performed using an enzyme-linked immunosorbent assay reader, and the absorbance was recorded. Using data processing software, with the concentration of α-KG as the abscissa and the absorbance measured by the enzyme-linked immunosorbent assay reader as the ordinate, linear regression was performed on the experimental data to obtain the α-KG standard curve.

[0101] The calculation formula for the molar formation rate is as follows:

[0102]

[0103] The results showed that BL21(DE3) / pET-28a-LAAD-ph3 could convert more than 0.6 mM of L-glutamic acid under the conditions of 30 °C and pH 7.0 for 24 h, and the molar yield of α-KG was 6.24%.

[0104] Similarly, referring to the method of this example, DH5α / pET-26b-LAAD-ph3 can convert more than 0.6 mM of L-glutamic acid under the conditions of 30 °C and pH 7.0 for 24 h, and the molar yield of α-KG is 6.07%. TOP10 / pET-28a-LAAD-ph3 can convert more than 0.6 mM of L-glutamic acid under the conditions of 30 °C and pH 7.0 for 24 h, and the molar yield of α-KG is 6.60%.

[0105] Example 5 Construction of L-amino acid deaminase mutant LAAD-phRK

[0106] The three-dimensional structure of LAAD-ph3 was predicted by Alphafold3, and the protein model LAAD-ph3 and L-glutamic acid were molecularly docked using the automatic docking software AutoDdck 4.0. By analyzing the key sites of the active center of the model and the interaction between the substrate L-glutamic acid and FAD and the enzyme, a total of 4 amino acid sites, namely ILE64 (I64), GLY257 (G257), GLN278 (Q278), and MET440 (M440), were selected for site-directed saturation mutagenesis. Using the amino acid sequence SEQ ID NO: 1 of L-amino acid deaminase LAAD-ph3 as a template, these 4 sites were mutated separately, and the corresponding coding genes were synthesized and transferred into Escherichia coli BL21(DE3) to construct a mutant engineering bacteria whole-cell catalyst, which was used to catalyze the synthesis of α-KG, and its yield was detected.

[0107] The construction of the whole-cell catalyst and the catalytic synthesis reaction were carried out under the following conditions:

[0108] Picked from the glycerol tube, streaked on solid LB medium (50 μg / mL kanamycin sulfate), and placed in a 37 °C incubator overnight. The next day, a single colony was picked and inoculated into 10 mL of LB liquid medium (50 μg / mL kanamycin sulfate), and cultured in a shaker at 37 °C and 180 rpm for 10 - 12 h to obtain a seed culture solution. The seed culture solution was transferred to TB medium (50 μg / mL kanamycin sulfate) at an inoculation amount of 5%, and cultured in a shaker at 37 °C and 180 rpm. When the OD 600 value reached 1.2, IPTG with a final concentration of 0.1 mM was added, and induced culture was carried out in a shaker at 16 °C and 180 rpm for 20 h. The fermentation broth after the induction culture was centrifuged to terminate fermentation, and the cells were collected. The centrifugation conditions were: 4 °C, 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 suspended with 5 mL of PBS buffer and stored at 4 °C for later use, that is, the L-amino acid deaminase mutant whole-cell catalyst was obtained.

[0109] The L-amino acid deaminase mutant whole-cell catalyst was applied to the synthesis of α-KG at 30 °C and pH 7.0. The reaction system was: 10 mM L-glutamic acid, 5 mM magnesium ions, and 1.5 g / L mutant whole-cell catalyst. The reaction was carried out with shaking at 180 rpm for 24 h, and the reaction was terminated by high-speed centrifugation for 5 min to remove the cells. The supernatant was collected, and the α-KG content was detected by a colorimetric method. The detection conditions were the same as those in Example 4.

[0110] The measurement results are as Figure 4 shown. The results showed that the catalytic activities of the mutants obtained by various mutation methods at these 4 mutation sites were significantly improved. Among them, the mutant LAAD-phRK (I64R / G257H / Q278R / M440K) had the highest α-KG production rate of 34.53% after recombinant expression, which was 4.53 times higher than that of the control group. Followed by I64R / Q278R / M440K, I64R / M440K, Q278R, Q278R / M440K, M440K, I64R, G257H, which were 27.63%, 26.13%, 26.02%, 17.81%, 17.73%, 16.91%, 10.08% respectively, which were 3.43 times, 3.19 times, 3.17 times, 1.85 times, 1.84 times, 1.71 times, 0.61 times higher than the control group.

[0111] The mutant LAAD-phRK was verified by sequencing, and its mutant amino acids were as follows: ILE (I) at position 64 was mutated to ARG (R), GLY (G) at position 257 was mutated to HIS (H), GLN (Q) at position 278 was mutated to ARG (R), and MET (M) at position 440 was mutated to LYS (K).

[0112] Among them, I64R: the codon was mutated from ATT to CGT; G257H: the codon was mutated from GGT to CAT; Q278R: the codon was mutated from CAG to CGT; M440K: the codon was mutated from ATG to AAG.

[0113] The amino acid sequence of the L-amino acid deaminase mutant LAAD-phRK obtained by screening is shown in SEQ ID NO: 3, and the optimized nucleotide sequence is shown in SEQ ID NO: 4. Among them, the amino acid sequence comparison diagram of L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK is as Figure 5 shown.

[0114] Similarly, according to the method of this embodiment, the molar yield of α-KG catalyzed by the mutant whole-cell catalyst obtained with Escherichia coli DH5α or TOP10 as the host is comparable to that of the mutant whole-cell catalyst obtained with Escherichia coli BL21(DE3) as the host.

[0115] Example 6 Synthesis of α-Ketoglutaric Acid Catalyzed by Mutant LAAD-phRK

[0116] The Escherichia coli strain BL21(DE3) recombinantly expressing the L-amino acid deaminase mutant LAAD-phRK was used to prepare the whole-cell catalyst under optimized conditions: picked from a glycerol tube, streaked on a solid LB medium (50 μg / mL kanamycin sulfate), and placed in an incubator at 37 °C for overnight culture. The next day, a single colony was picked and inoculated into 10 mL of LB liquid medium (50 μg / mL kanamycin sulfate), and cultured in a shaker at 37 °C and 180 rpm for 10 - 12 h to obtain a seed culture solution. The seed culture solution was transferred to TB medium (50 μg / mL kanamycin sulfate) at an inoculation amount of 5%, and cultured in a shaker at 37 °C and 180 rpm. When the OD 600 value reached 1.0, IPTG with a final concentration of 0.1 mM was added, and induced culture was carried out in a shaker at 16 °C and 180 rpm for 16 h. The fermentation broth after the induced culture was centrifuged to terminate fermentation, and the cells were collected. The centrifugation conditions were: 4 °C, 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 suspended in 5 mL of PBS buffer and stored at 4 °C for later use, thus obtaining the L-amino acid deaminase mutant LAAD-phRK whole-cell catalyst.

[0117] Optimized catalytic synthesis reaction conditions: The L-amino acid deaminase mutant LAAD-phRK whole-cell catalyst was applied to the synthesis of α-KG at 30 °C and pH 8.0. The reaction system was: 10 mM L-glutamic acid, 10 mM calcium ion, and 1.5 g / L of the mutant LAAD-phRK whole-cell catalyst. The reaction was carried out with shaking at 180 rpm for 8 h, and the reaction was terminated by high-speed centrifugation for 5 min to remove the cells. The supernatant was collected, and the α-KG content was detected by the colorimetric method. The detection conditions were the same as those in Example 4. This recombinant Escherichia coli mutant strain LAAD-phRK can convert more than 6 mM of L-glutamic acid under the conditions of 30 °C and pH 8.0 for 8 h, and the molar yield of α-KG is 64.89%.

[0118] Similarly, following the method of this example, DH5α / pET-26b-LAAD-phRK can convert more than 6 mM of L-glutamic acid with a molar yield of α-KG of 60.84% when reacting at 30 °C and pH 8.0 for 8 h. TOP10 / pET-28a-LAAD-phRK can convert more than 6 mM of L-glutamic acid with a molar yield of α-KG of 68.12% when reacting at 30 °C and pH 8.0 for 8 h.

[0119] In summary, the L-amino acid deaminase LAAD of the present invention can stably and efficiently convert L-glutamic acid to generate α-ketoglutaric acid.

[0120] The above experimental results can further illustrate that the present invention can be used for actual production safely and efficiently, thereby effectively reducing the input of actual costs.

[0121] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. L - amino acid deaminase mutant, characterized in that: The amino acid sequence of the mutant is obtained by mutating SEQ ID NO: 1 in any of the following ways: Q278R, Q278R / M440K, I64R / Q278R / M440K, or I64R / G257H / Q278R / M440K; wherein, the amino acid sequence of the mutant I64R / G257H / Q278R / M440K is as shown in SEQ ID NO:

3.

2. A gene encoding the L-amino acid deaminase mutant according to claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the mutant I64R / G257H / Q278R / M440K is as shown in SEQ ID NO:

4.

4. The biological material related to the L-amino acid deaminase mutant according to claim 1, characterized in that: is any one or a combination of the following biological materials: (a) An expression cassette containing the gene according to claim 2 or 3; (b) A recombinant expression vector containing the gene according to claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant microorganism containing the gene according to claim 2 or 3; (e) A recombinant microorganism containing the expression cassette described in (a); (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c); The host microorganism corresponding to the recombinant microorganism described in (d), (e), or (f) is selected from prokaryotes.

5. The biological material according to claim 4, characterized in that: The starting vector of the recombinant expression vectors described in (b) and (c) is a vector of the pET series.

6. Use of the gene according to any one of claims 2 to 3 or the biological material according to any one of claims 4 to 5 in the preparation of an L-amino acid deaminase mutant.

7. Use of the L-amino acid deaminase mutant according to claim 1, the gene according to any one of claims 2 to 3, the biological material according to any one of claims 4 to 5, the L-amino acid deaminase LAAD-ph3, or the biological material related to the L-amino acid deaminase LAAD-ph3 in the biosynthesis of α-ketoglutaric acid, characterized in that: The amino acid sequence of the L-amino acid deaminase LAAD-ph3 is as shown in SEQ ID NO: 1; The biological material related to the L-amino acid deaminase LAAD-ph3 is any one or a combination of the following biological materials: 1) An expression cassette containing the gene encoding the L-amino acid deaminase LAAD-ph3; 2) A recombinant expression vector containing the gene encoding the L-amino acid deaminase LAAD-ph3; 3) A recombinant expression vector containing the expression cassette described in 1); 4) A recombinant microorganism containing the gene encoding the L-amino acid deaminase LAAD-ph3; 5) A recombinant microorganism containing the expression cassette described in 1); 6) A recombinant microorganism containing the recombinant expression vector described in 2) or 3); The host microorganism corresponding to the recombinant microorganism described in 4), 5), or 6) is selected from prokaryotes.

8. A method for obtaining the L-amino acid deaminase mutant according to claim 1, characterized in that, It includes the following steps: The gene of L-amino acid deaminase LAAD-ph3 encoding the amino acid sequence shown in SEQ ID NO: 1 is subjected to site-directed mutagenesis by site-directed saturation mutagenesis technology and then expressed to obtain the L-amino acid deaminase mutant described in claim 1.

9. A method for producing α-ketoglutaric acid, characterized in that, It includes the following steps: Using the recombinant microorganism described in any one of claims 4 to 5 or the recombinant microorganism described in claim 7 as a whole-cell catalyst, a substrate containing L-glutamic acid is transformed to obtain α-ketoglutaric acid.

10. The method according to claim 9, wherein: A reaction system containing 1.5 - 2.0 g / L whole cells, 10 - 15 mM L-glutamic acid, and 5 - 10 mM magnesium ions or calcium ions is reacted at 30 - 35 °C and pH 7.0 - 8.0 for 8 - 24 h.

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