L-amino acid deaminase as well as mutant and application thereof

Abstract

The invention discloses L-amino acid deaminase as well as a 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 yield of alpha-KG generated through catalysis of the mutant LAAD-phRK is increased by 4.53 times. 1.5 g / L of LAAD-phRK whole cells react in a reaction system containing 10 mM of L-glutamic acid and 10 mM of calcium ions at the temperature of 30 DEG C and the pH value of 8.0 for 8 h, L-glutamic acid with the concentration of 6 mM or above can be converted, the molar yield of alpha-KG is 60% or above, and the LAAD-phRK whole cells have wide prospects in large-scale industrial application.

Description

Technical Field

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

[0002] α-ketoglutarate (α-KG) is one of the important intermediates of the tricarboxylic acid cycle and participates 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. It 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 hydroxylated products such as 4-hydroxyisoleucine (4-HIL) generated by α-ketoglutarate-dependent hydroxylase. So far, α-ketoglutarate and its derivatives have been widely used in many fields such as chemical industry, food, medicine, daily chemicals, etc., and have very broad application prospects.

[0003] At present, the synthesis methods of α-ketoglutaric acid mainly include chemical method, microbial fermentation method and enzyme catalysis method. The chemical method for producing α-ketoglutaric acid usually uses diethyl succinate, diethyl oxalate and the like as raw materials, and synthesizes α-ketoglutaric acid through condensation, hydrolysis, distillation concentration, crystallization and other processes, with a maximum yield of 75%. However, the starting raw materials for the chemical method of producing α-KG have a special structure, contain toxic substances that pollute the environment, require multiple steps of reaction, have a low final yield, and have serious safety issues, which limits its application in industries such as food and cosmetics. For decades, the production of α-KG by microbial fermentation has been continuously developing and has made great progress, but there are also unfavorable factors such as the lack of production strain resources, low metabolite yield, difficult separation and long production cycle, which cannot meet the needs of large-scale industrial production. The enzyme catalytic conversion method has the advantages of mild reaction conditions, single product, simple extraction process, high product concentration and high purification yield, so the enzyme catalytic method for producing α-ketoglutaric acid has become the mainstream. There are three main pathways for the enzyme-catalyzed synthesis of α-ketoglutarate, all of which use L-glutamate as raw material and involve glutamate dehydrogenase (GDH), L-amino acid oxidase (AAO, EC 1.4.3.2 and EC1.4.3.3) and L-amino acid deaminase (LAD, EC 1.4.3.2) to catalyze the synthesis of α-ketoglutarate.

[0004] The above pathway 1 is because glutamate dehydrogenase has a higher affinity for α-ketoglutarate than glutamate and the reaction requires the coenzyme nicotinamide adenine dinucleotide phosphate (NADP). + ) are involved, so the yield of α-ketoglutarate is extremely low. The L-amino acid oxidase in pathway two will produce hydrogen peroxide during the reaction, which seriously inhibits the activity of the enzyme. Although the addition of catalase (CAT) can eliminate hydrogen peroxide, it is difficult to be applied industrially due to the complexity of the production process and increased production costs. The L-amino acid deaminase in pathway three also catalyzes the synthesis of α-ketoglutarate using L-glutamate as a substrate. The reaction process only produces α-ketoglutarate, ammonia and water. There is no production of toxic substances such as H2O2, which has little effect on cell growth. In comparison, it is more conducive to the production of α-ketoglutarate. Current research has known that the enzyme derived from Proteus mirabilis ( Proteus mirabilis ) can be heterologously expressed in Escherichia coli BL21 (DE3) and effectively convert L-glutamate into α-ketoglutarate. However, as a membrane protein, pm1 has a low expression level and a low specificity for the substrate L-glutamate (35.8%). Currently, there are few studies on L-amino acid deaminases, and very few types of L-amino acid deaminases that can effectively catalyze the synthesis of α-ketoglutarate have been discovered. The only reported one is from Proteus mirabilis The presence of pm1 in the enzyme-catalyzed synthesis of α-ketoglutarate has limited its application in industry. Therefore, discovering new L-amino acid deaminases with high efficiency and improving their enzyme activity through directed evolution will help to further reduce the cost of industrial production. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art α-ketoglutaric acid synthesis process, the primary purpose of the present invention is to provide a Proteus hauseri L-amino acid deaminase and its mutants.

[0006] Another object of the present invention is to provide a gene encoding the above-mentioned L-amino acid deaminase and its mutants.

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

[0008] The present invention verifies for the first time that Proteus housiei ( Proteus hauseri) has the ability to catalyze L-glutamate to produce α-ketoglutarate. Further, through protein three-dimensional structure simulation, molecular docking, and virtual saturation mutation, it is predicted that the related sites that may affect catalysis and binding are ILE64, GLY257, GLN278, and MET440. Site-directed saturation mutations are performed on these four sites to obtain mutants and engineered bacteria that efficiently catalyze the synthesis of α-ketoglutarate, which has good application prospects.

[0009] The purpose of the present invention is achieved through the following technical solutions: An L-amino acid deaminase mutant, wherein the amino acid sequence of SEQ ID NO: 1 is obtained by any one of the following mutations: At least one of I64R, G257H, Q278R and M440K; among them, I64R, i.e. the 64th amino acid mutates from ILE (I) to ARG (R), and the same applies to the others.

[0010] Furthermore, the L-amino acid deaminase mutant has an amino acid sequence of SEQ ID NO: 1 obtained by any of the following mutations: I64R, G257H, Q278R, M440K, I64R / M440K, Q278R / M440K, I64R / Q278R / M440K, or I64R / G257H / Q278R / M440K.

[0011] Preferably, the gene sequence encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2.

[0012] A gene encoding the L-amino acid deaminase mutant.

[0013] Preferably, an L-amino acid deaminase mutant LAAD-phRK (I64R / G257H / Q278R / M440K), whose amino acid sequence is shown in SEQ ID NO: 3.

[0014] A gene encoding the L-amino acid deaminase mutant LAAD-phRK, whose nucleotide sequence is shown in SEQ ID NO: 4.

[0015] The mutant-related biological material is any one or more combinations of the following biological materials: (a) an expression cassette containing the above-mentioned coding gene; (b) a recombinant expression vector containing the above encoding gene; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the above encoding gene; (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).

[0016] Furthermore, the starting vector of the recombinant expression vector 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.

[0017] Furthermore, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes or yeast, etc.; the prokaryotes include Escherichia ( Escherichia ) and other bacteria; the yeast includes yeast such as Pichia pastoris. More specifically, the prokaryotic organism 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.

[0018] Application of the mutant, encoding gene and mutant-related biological materials in the preparation of L-amino acid deaminase mutants.

[0019] Furthermore, the application of the mutant, the encoding gene, the mutant-related biomaterial, L-amino acid deaminase LAAD-ph3, and the L-amino acid deaminase LAAD-ph3-related biomaterial is one of the following applications: (I) Application in the biosynthesis of α-ketoglutaric acid; (II) Application in food, pharmaceuticals, feed, cosmetics and chemicals; (III) Application in health care products.

[0020] The L-amino acid deaminase LAAD-ph3 related biomaterial is any one or more combinations of the following biomaterials: 1) an expression cassette containing a gene encoding L-amino acid deaminase LAAD-ph3; 2) a recombinant expression vector containing a gene encoding L-amino acid deaminase LAAD-ph3; 3) a recombinant expression vector containing the expression cassette described in 1); 4) A recombinant microorganism containing a gene encoding 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).

[0021] Furthermore, the starting vector of the recombinant expression vector in 2) and 3) is a pET series vector or a pPICZα vector, etc.; preferably a pET-28a(+) vector, a pET-26b(+) or a pPICZαA vector.

[0022] Furthermore, the host microorganism corresponding to the recombinant microorganism in 4), 5), and 6) is selected from prokaryotes or yeast, etc.; the prokaryotes include Escherichia ( Escherichia ) and other bacteria; the yeast includes yeast such as Pichia pastoris. More specifically, the prokaryotic organism 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.

[0023] Wherein, the L-amino acid deaminase LAAD-ph3 is derived from Proteus hauseri , whose amino acid sequence is shown in SEQ ID NO: 1; and whose nucleotide sequence of the gene encoding L-amino acid deaminase LAAD-ph3 is shown in SEQ ID NO: 2.

[0024] A method for obtaining the above mutant comprises the following steps: performing site-directed mutagenesis on a gene encoding an L-amino acid deaminase LAAD-ph3 having an amino acid sequence as shown in SEQ ID NO: 1 by site-directed saturation mutagenesis technology and then expressing the site-directed mutagenesis to obtain an L-amino acid deaminase mutant.

[0025] Furthermore, mutations were introduced into the gene encoding the L-amino acid deaminase LAAD-ph3 having an amino acid sequence as shown in SEQ ID NO: 1 by site-directed saturation mutagenesis technology, and after correct sequencing, the gene was transformed into Escherichia coli for expression to obtain an L-amino acid deaminase mutant.

[0026] Preferably, the Escherichia coli includes Escherichia coli BL21 (DE3), Escherichia coli DH5α or Escherichia coli TOP10 and the like.

[0027] A method for producing α-ketoglutaric acid comprises the following steps: The recombinant microorganism in the biomaterial related to the above mutant or the recombinant microorganism in the biomaterial related to L-amino acid deaminase LAAD-ph3 is used as a whole-cell catalyst to convert the substrate containing L-glutamate to obtain α-ketoglutarate.

[0028] Furthermore, a reaction system containing 1.5-2.0 g / L whole cells, 10-15 mM L-glutamate, 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.

[0029] Furthermore, a reaction system containing 1.5 g / L L-amino acid deaminase LAAD-ph3 whole cells, 10 mM L-glutamate, and 5 mM magnesium ions was reacted at 30°C and pH 7.0 for 24 h; more than 0.6 mM L-glutamate was converted, and the molar yield of α-KG was above 6%.

[0030] Furthermore, a reaction system containing 1.5 g / L L-amino acid deaminase mutant LAAD-phRK whole cells, 10 mM L-glutamate, and 10 mM calcium ions was reacted at 30°C and pH 8.0 for 8 h; more than 6 mM L-glutamate could be converted, and the molar yield of α-KG was above 60%.

[0031] The whole cells are collected as follows: the seed culture solution of the recombinant microorganism in the above mutant-related biological material or the recombinant microorganism in the L-amino acid deaminase LAAD-ph3-related biological material is transferred to the fermentation medium at an inoculation rate of 4-6% (or further 5%), and fermented until the OD 600 When the value was 0.8-1.4, IPTG was added at a final concentration of 0.1 mM to induce the culture and collect the cells.

[0032] Preferably, the seed culture solution is prepared in the following manner: a single colony of the recombinant microorganism in the above-mentioned mutant-related biological material or the recombinant microorganism in the L-amino acid deaminase LAAD-ph3-related biological material is inoculated into LB liquid culture medium and cultured with shaking to obtain a seed culture solution.

[0033] The shaking culture conditions are 37±1° C. and 180±20 rpm for 10 to 12 h.

[0034] Preferably, the fermentation medium comprises TB medium.

[0035] Preferably, the induction culture is carried out at 16±1°C and 180±20 rpm in a shaking incubator for 16 to 20 h.

[0036] 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.

[0037] Compared with the prior art, the present invention has the following advantages and effects: (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%.

[0038] 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.

[0039] 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.

[0040] (2) The present invention obtained 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 catalyzing L-glutamate to produce α-ketoglutarate was significantly improved. 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 catalytic production of α-ketoglutarate is increased by 4.53 times. The amino acid sequence of the L-amino acid deaminase mutant LAAD-phRK has not been reported in domestic and foreign literature before.

[0041] (3) The present invention provides Proteus hauseri The L-amino acid deaminase mutant LAAD-phRK from the source has the ability to efficiently catalyze L-glutamate to produce α-ketoglutarate. 1.5 g / L LAAD-phRK whole-cell catalyst can convert more than 6 mM L-glutamate in a reaction system of 10 mM L-glutamate and 10 mM calcium ions at 30°C and pH 8.0 for 8 h, and the molar yield of α-KG is above 60%.

[0042] In summary, the present invention Proteus hauseri The L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK derived from this plant can efficiently synthesize α-ketoglutarate and have broad prospects for large-scale industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure is the SDS-PAGE electrophoresis result of the recombinant Escherichia coli BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK in Example 1; wherein, 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.

[0044] Figure 2 This is the SDS-PAGE electrophoresis result of the 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.

[0045] Figure 3 This is the SDS-PAGE electrophoresis result of the 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.

[0046] Figure 4This is a graph showing the determination of the molar production rate of α-KG produced by the recombinant Escherichia coli expressing the LAAD-ph3 mutant enzyme in Example 5.

[0047] Figure 5 It is a comparison chart of the amino acid sequences of L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0049] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0050] Example 1 Construction of L-amino acid deaminase LAAD-ph3 and LAAD-phRK encoding genes transferred into Escherichia coli BL21 (DE3) genetically engineered bacteria and preparation of whole-cell catalyst We commissioned Shenggong Bioengineering Co., Ltd. to synthesize the full-length gene encoding the corresponding amino acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4). NcoI / XOt The corresponding enzyme digestion was performed and connected to the pET-28a(+) plasmid digested with the same enzyme digestion. The 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 transformed into Escherichia coli BL21(DE3) competent cells to construct the engineering bacteria BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK.

[0051] The engineered bacteria BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK were inoculated into 10 mL LB liquid medium (50 μg / mL kanamycin sulfate) and cultured at 37°C and 180 rpm for 10-12 h to obtain seed culture solution. The seed culture solution was transferred to TB medium (50 μg / mL kanamycin sulfate) at a 5% inoculation rate and cultured at 37°C and 180 rpm. When the bacteria grew to OD 600When the value is 0.8-1.4, add IPTG with a final concentration of 0.1 mM, and induce culture in a shaker at 16 ° C and 180 rpm for 20 h. After the induction culture, the fermentation broth is centrifuged to stop the fermentation and collect the bacteria. The centrifugation conditions are: 4 ° C, 8000 rpm, 10 min. After centrifugation, wash the cells with 5 mL PBS buffer, centrifuge again, and repeat this step once. Finally, suspend the bacteria with 5 mL PBS buffer and place at 4 ° C for use, and obtain L-amino acid deaminase BL21 (DE3)-pET-28a-LAAD-ph3 and BL21 (DE3)-pET-28a-LAAD-phRK whole cell catalysts.

[0052] The BL21(DE3) / pET-28a-LAAD-ph3 and BL21(DE3) / pET-28a-LAAD-phRK whole cell catalysts were analyzed by gel electrophoresis using 12% (W / V) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE.

[0053] The conditions for gel electrophoresis are as follows: Sample treatment: Take 10 μL of loading buffer (5x), mix 40 μL of sample with loading buffer (5x) in a ratio of 4:1, and boil (95°C) for 5 min; Sample loading volume: 10 μL, Maker loading volume: 5 μL; Voltage: 150 mV, current: 90 mA, duration: 90 min.

[0054] 1x electrode buffer (pH 8.3): 14.4 g glycine, 3 g Tris, 1 g SDS, dissolve in distilled water and make up to 1 L; 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 500 mL; Decolorizing solution (1 L): 100 mL methanol, 100 mL acetic acid, add distilled water to make up to 1 L.

[0055] The electrophoresis results are 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 lane 2 of the control group, an obvious protein band (52 kDa position) appeared in the protein expressed by the engineering bacteria.

[0056] Example 2 Construction of L-amino acid deaminase LAAD-ph3 and LAAD-phRK encoding genes transferred into Escherichia coli DH5α genetically engineered bacteria and preparation of whole-cell catalyst We commissioned Shenggong Bioengineering Co., Ltd. to synthesize the full-length gene encoding the corresponding amino acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4). NcoI / XOt The corresponding enzyme digestion was performed and connected to the pET-26b(+) plasmid with the same enzyme digestion. The positive transformants were screened by sequencing verification to obtain the positive recombinant plasmids pET-26b-LAAD-ph3 and pET-26b-LAAD-phRK. The constructed plasmids were transformed into Escherichia coli DH5α competent cells to construct the engineered bacteria DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK.

[0057] The L-amino acid deaminase DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK whole cell catalysts were prepared using the same method as in Example 1.

[0058] The DH5α / pET-26b-LAAD-ph3 and DH5α / pET-26b-LAAD-phRK whole cell catalysts were analyzed by gel electrophoresis using 12% (W / V) sodium dodecyl sulfate polyacrylamide gel SDS-PAGE, using the same method as in Example 1.

[0059] The electrophoresis results are 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 lane 1 of the control group, an obvious protein band (52 kDa position) appeared in the protein expressed by the engineering bacteria.

[0060] Example 3 Construction of L-amino acid deaminase LAAD-ph3 and LAAD-phRK encoding genes transferred into Escherichia coli TOP10 genetically engineered bacteria and preparation of whole-cell catalyst We commissioned Shenggong Bioengineering Co., Ltd. to synthesize the full-length gene encoding the corresponding amino acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 3 (as shown in SEQ ID NO: 2 and SEQ ID NO: 4). N / XOtThe corresponding enzyme digestion was performed and connected to the pET-28a(+) plasmid digested with the same enzyme digestion. The 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 transformed into Escherichia coli TOP10 competent cells to construct the engineered bacteria TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK.

[0061] The L-amino acid deaminase TOP10 / pET-28a-LAAD-ph3 and TOP10 / pET-28a-LAAD-phRK whole cell catalysts were prepared using the same method as in Example 1.

[0062] 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, using the same method as in Example 1.

[0063] The electrophoresis results are as follows Figure 3 As 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 lane 2 of the control group, an obvious protein band (52 kDa position) appeared in the protein expressed by the engineering bacteria.

[0064] Example 4: Recombinant expression of L-amino acid deaminase LAAD-ph3 to catalyze the synthesis of α-ketoglutarate The prepared 1.5 g / L recombinantly expressed BL21(DE3) / pET-28a-LAAD-ph3 whole-cell catalyst was placed in a reaction system of 10 mM L-glutamate and 5 mM magnesium ions at 30°C and pH 7.0 for 24 h with oscillation.

[0065] The molar yield of α-KG synthesized from L-Glu was determined by the BL21(DE3) / pET-28a-LAAD-ph3 whole cell catalyst.

[0066] The method for determining the molar production rate of α-KG is as follows: Reaction system: 10 mM L-glutamate (L-Glu), 5 mM magnesium ion and 1.5 g / L whole cell catalyst. Under the conditions of 30℃ and pH 7.0, the reaction was shaken for 24 h, and the bacteria were removed by high-speed centrifugation for 5 min to terminate the reaction. The supernatant was collected and the α-KG content was detected by colorimetric method.

[0067] The method for detecting α-KG content by colorimetric method is as follows: Take 50 μL of supernatant and mix it with 100 μL of 0.1% 2,4-dinitrophenylhydrazine, let it stand at room temperature for 5 min, then add 1 mL of 1.5 M sodium hydroxide solution. After the color stabilizes, pipette 200 μL of liquid into a 96-well shallow well plate and place it in an ELISA reader to detect its absorbance at a wavelength of 520 nm.

[0068] The method for establishing the α-KG standard curve is as follows: Use α-KG standard to prepare 6 groups of α-KG standard solutions of 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM, respectively. Use enzyme-linked immunosorbent assay to detect and record the absorbance. Use data processing software to perform linear regression on the experimental data with the concentration of α-KG as the horizontal axis and the absorbance measured by the enzyme-linked immunosorbent assay as the vertical axis to obtain the standard curve of α-KG.

[0069] The molar generation rate is calculated as follows:

[0070] The results showed that BL21(DE3) / pET-28a-LAAD-ph3 could convert more than 0.6 mM L-glutamate at 30°C and pH 7.0 for 24 h, and the molar yield of α-KG was 6.24%.

[0071] Similarly, referring to the method of this embodiment, DH5α / pET-26b-LAAD-ph3 can convert more than 0.6 mM of L-glutamic acid at 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 at 30°C and pH 7.0 for 24 h, and the molar yield of α-KG is 6.60%.

[0072] Example 5 Construction of L-amino acid deaminase mutant LAAD-phRK Alphafold3 was used to predict the three-dimensional structure of LAAD-ph3, and the automatic docking software AutoDdck 4.0 was used to perform molecular docking between the protein model LAAD-ph3 and L-glutamic acid. By analyzing the key sites of the model's active center and the interaction between the substrates L-glutamic acid and FAD and the enzyme, four amino acid sites, 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 four sites were mutated respectively, and the corresponding coding genes were synthesized and transferred into Escherichia coli BL21 (DE3) to construct a mutant engineered bacteria whole-cell catalyst, which was used to catalyze the synthesis of α-KG and its yield was detected.

[0073] The following conditions were used to construct the whole-cell catalyst and catalyze the synthesis reaction: Pick out from the glycerol tube, streak on solid LB medium (50 μg / mL kanamycin sulfate), and place in a 37 ℃ incubator overnight. The next day, pick a single colony and inoculate it in 10 mL LB liquid medium (50 μg / mL kanamycin sulfate), and culture it in a shaker at 37℃ and 180 rpm for 10-12 h to obtain seed culture solution. Transfer the seed culture solution to TB medium (50 μg / mL kanamycin sulfate) at a 5% inoculation rate and culture it in a shaker at 37℃ and 180 rpm. When the bacteria grow to OD 600 When the value is 1.2, add IPTG with a final concentration of 0.1 mM, and induce culture in a shaker at 16°C and 180 rpm for 20 h. After the induction culture, the fermentation broth is centrifuged to stop the fermentation and collect the bacteria. The centrifugation conditions are: 4°C, 8000 rpm, 10 min. After centrifugation, wash the cells with 5 mL PBS buffer, centrifuge again, and repeat this step once. Finally, suspend the bacteria with 5 mL PBS buffer and place at 4°C for use, and the L-amino acid deaminase mutant whole-cell catalyst is obtained.

[0074] 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 was: 10 mM L-glutamate, 5 mM magnesium ions and 1.5 g / L mutant whole cell catalyst. The reaction was shaken at 180 rpm for 24 h, and the bacteria were removed by high-speed centrifugation for 5 min to terminate the reaction. The supernatant was collected and the α-KG content was detected by colorimetric method. The detection conditions were consistent with those in Example 4.

[0075] The results of the test are as follows Figure 4As shown, the results showed that the catalytic activity of the mutants obtained by various mutations of these four mutation sites was significantly improved. Among them, the mutant LAAD-phRK (I64R / G257H / Q278R / M440K) had the highest α-KG yield 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%, and 10.08%, respectively, which were 3.43 times, 3.19 times, 3.17 times, 1.85 times, 1.84 times, 1.71 times, and 0.61 times higher than that of the control group.

[0076] The mutant LAAD-phRK was verified by sequencing, and its mutated amino acids were as follows: ILE (I) at position 64 mutated to ARG (R), GLY (G) at position 257 mutated to HIS (H), GLN (Q) at position 278 mutated to ARG (R), and MET (M) at position 440 mutated to LYS (K).

[0077] Among them, I64R: the codon mutated from ATT to CGT; G257H: the codon mutated from GGT to CAT; Q278R: the codon mutated from CAG to CGT; M440K: the codon mutated from ATG to AAG.

[0078] 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 of L-amino acid deaminase LAAD-ph3 and its mutant LAAD-phRK is shown in Figure 5 shown.

[0079] Similarly, referring to the method of this embodiment, the molar yield of α-KG synthesized by the mutant whole-cell catalyst obtained with Escherichia coli DH5α or TOP10 as the host is equivalent to the molar yield of α-KG synthesized by the mutant whole-cell catalyst obtained with Escherichia coli BL21 (DE3) as the host.

[0080] Example 6 Synthesis of α-ketoglutarate catalyzed by mutant LAAD-phRK The Escherichia coli strain BL21 (DE3) expressing the recombinant L-amino acid deaminase mutant LAAD-phRK was used to prepare the whole-cell catalyst under the optimized conditions: the whole cell was picked out from the glycerol tube, streaked on solid LB medium (50 μg / mL of kanamycin sulfate), and placed in a 37°C incubator for overnight culture. The next day, a single colony was picked and inoculated into 10 mL of LB liquid medium (50 μg / mL of kanamycin sulfate), and cultured in a shaker at 37°C and 180 rpm for 10-12 h to obtain the seed culture solution. The seed culture solution was transferred to TB medium (50 μg / mL of kanamycin sulfate) at an inoculation rate of 5%, and cultured in a shaker at 37°C and 180 rpm. When the bacteria grew to OD 600 When the value is 1.0, add IPTG with a final concentration of 0.1 mM, and induce culture in a shaker at 16°C and 180 rpm for 16 h. After the induction culture, the fermentation broth is centrifuged to stop the fermentation and collect the bacteria. The centrifugation conditions are: 4°C, 8000rpm, 10 min. After centrifugation, wash the cells with 5 mL PBS buffer, centrifuge again, and repeat this step once. Finally, suspend the bacteria with 5 mL PBS buffer and place at 4°C for use, and the L-amino acid deaminase mutant LAAD-phRK whole cell catalyst is obtained.

[0081] 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°C and pH 8.0. The reaction system was: 10 mM L-glutamate, 10 mM calcium ions and 1.5 g / L mutant LAAD-phRK whole cell catalyst. The reaction was oscillated at 180 rpm for 8 h, and the bacteria were removed by high-speed centrifugation for 5 min to terminate the reaction. The supernatant was collected and the α-KG content was detected by colorimetric method. The detection conditions were consistent with those in Example 4. The recombinant Escherichia coli mutant strain LAAD-phRK was reacted for 8 h at 30°C and pH 8.0, and more than 6 mM L-glutamate could be converted, and the molar yield of α-KG was 64.89%.

[0082] Similarly, referring to the method of this example, DH5α / pET-26b-LAAD-phRK can convert more than 6 mM of L-glutamate at 30°C and pH 8.0 for 8 h, and the molar yield of α-KG is 60.84%. TOP10 / pET-28a-LAAD-phRK can convert more than 6 mM of L-glutamate at 30°C and pH 8.0 for 8 h, and the molar yield of α-KG is 68.12%.

[0083] In summary, the L-amino acid deaminase LAAD of the present invention can stably and efficiently convert L-glutamate into α-ketoglutarate.

[0084] The above experimental results can further illustrate that the present invention can be safely and efficiently used in actual production, thereby effectively reducing the actual cost investment.

[0085] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

4.

4. The biomaterial related to the L-amino acid deaminase mutant according to claim 1, characterized in that: Any one or more combinations 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).

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

6. Use of the L-amino acid deaminase mutant according to claim 1, the gene according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 5 in the preparation of the 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 biomaterial according to any one of claims 4 to 5, L-amino acid deaminase LAAD-ph3 or L-amino acid deaminase LAAD-ph3 related biomaterials, characterized in that: One of the following applications: (I) Application in the biosynthesis of α-ketoglutaric acid; (II) Application in food, pharmaceuticals, feed, cosmetics and chemicals; (III) Application in health care products; The amino acid sequence of the L-amino acid deaminase LAAD-ph3 is shown in SEQ ID NO: 1; The L-amino acid deaminase LAAD-ph3 related biomaterial is any one or more combinations of the following biomaterials: 1) an expression cassette containing a gene encoding L-amino acid deaminase LAAD-ph3; 2) a recombinant expression vector containing a gene encoding L-amino acid deaminase LAAD-ph3; 3) a recombinant expression vector containing the expression cassette described in 1); 4) A recombinant microorganism containing a gene encoding 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).

8. A method for obtaining the L-amino acid deaminase mutant according to claim 1, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on a gene encoding an L-amino acid deaminase LAAD-ph3 having an amino acid sequence as shown in SEQ ID NO: 1 by site-directed saturation mutagenesis technology and then expressing the site-directed mutagenesis to obtain the L-amino acid deaminase mutant according to claim 1.

9. A method for producing α-ketoglutaric acid, characterized in that: The steps include: The recombinant microorganism described in any one of claims 4 to 5 or the recombinant microorganism described in claim 7 is used as a whole-cell catalyst to convert a substrate containing L-glutamate to obtain α-ketoglutarate.

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

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