D-amino acid oxidase mutant and application thereof

Abstract

The invention discloses a D-amino acid oxidase mutant and application thereof. Based on the amino acid sequence of wild type D-amino acid oxidase, the amino acid sequence of the mutant comprises substitution of V53F and / or Y242V, and the amino acid sequence of the wild type D-amino acid oxidase comprises a sequence as shown in SEQ ID No.1. The D-amino acid oxidase mutant disclosed by the invention has high catalytic activity and high stability.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a D-amino acid oxidase mutant and application thereof. Background Art

[0002] Due to their structural diversity and rich functionality, short-chain unnatural amino acids are widely used in fields such as medicine, chemical engineering, and agriculture. However, their synthesis is relatively difficult, and the modification of key enzymes in their synthesis process and the development of catalytic processes have important theoretical value and potential economic value. The target products of the present invention are L-norvaline and L-2-aminobutyric acid. L-norvaline is used as a key intermediate in the synthesis of perindopril, a common hypertension treatment drug, and L-2-aminobutyric acid is commonly used in the synthesis of the anti-tuberculosis drug ethambutol and the anti-epileptic drugs levetiracetam and brivaracetam. Methods for synthesizing these short-chain unnatural amino acids include chemical methods and biocatalytic methods. Chemical methods generally have disadvantages such as low optical purity, harsh reaction conditions, and cumbersome steps. Compared with chemical synthesis methods, biocatalytic methods can economically, greenly, and efficiently synthesize L-norvaline and L-2-aminobutyric acid of high chiral purity, with significant social benefits and potential commercial value.

[0003] D-amino acid oxidase (DAAO, EC 1.4.3.3) uses flavin adenine dinucleotide (FAD) as a cofactor and belongs to the typical flavoproteinase class. Under aerobic conditions, it selectively catalyzes the oxidative dehydrogenation of D-amino acids to form the corresponding α-imino acids. The resulting imino acids spontaneously hydrolyze to α-keto acids and ammonia. Simultaneously, oxygen molecules reoxidize the reduced FAD to generate H2O2. D-amino acid oxidase can be used in a cascade with catalase, amino acid dehydrogenase, and formate dehydrogenase to produce L-short-chain unnatural amino acids using a four-enzyme, one-pot process. The main reaction process involves the initial oxidation of D-amino acids in a mixed substrate to keto acids by DAAO. Catalase then removes the H2O2 byproduct. The cascade is followed by amino acid dehydrogenase, followed by formate dehydrogenase, which then undergoes a coenzyme cycle to convert the keto acids into L-short-chain unnatural amino acids. This deracemization reaction produces L-short-chain unnatural amino acids.

[0004] Currently reported D-amino acid oxidases targeting the substrates D-norvaline and D-2-aminobutyric acid generally suffer from low activity, moderate thermal stability, and poor solubility in E. coli expression systems. Therefore, there is a need to develop D-amino acid oxidase catalysts with significantly improved industrial applicability to further increase the yield and conversion rate of the target products. Summary of the Invention

[0005] In view of the problems of low catalytic activity, poor thermal stability and poor soluble expression of D-amino acid oxidase in the prior art, the present invention provides a D-amino acid oxidase mutant and its application.

[0006] The present invention first screened the DAAO enzyme library to obtain a high-activity enzyme from Fusarium graminearum ( Fusarium graminearum D-amino acid oxidase Fg DAAO. Then from Fg Starting from DAAO, the gene was modified by site-directed mutagenesis to improve Fg The catalytic activity and stability of DAAO were enhanced to achieve its soluble expression in Escherichia coli to further increase the production rate of 2-ketopentanoic acid and 2-ketobutyric acid, and lay the foundation for more efficient multi-enzyme cascade industrial production of L-short-chain unnatural amino acids.

[0007] To solve Fg The problem of poor soluble expression and stability of DAAO is addressed by the present invention through the ESM model. Fg Single-point mutation prediction using the DAAO sequence identified the V53F mutation, which significantly improved thermostability. Residual enzyme activity reached 95% after 15 minutes at 60°C for the substrate D,L-norvaline, a 6.3-fold increase compared to the wild-type residual activity of 15%. The half-life at 60°C increased from 4.9 minutes in the wild-type to 127 minutes, a 25-fold increase. Residual enzyme activity reached 99% after 15 minutes at 60°C for the substrate D,L-2-aminobutyric acid, a 9.0-fold increase compared to the wild-type residual activity of 11%. The half-life at 60°C increased from 5.4 minutes in the wild-type to 189 minutes, a 35-fold increase. V53F also significantly improved the relative activities of the two substrates: 2.1-fold for D,L-norvaline and 1.6-fold for D,L-2-aminobutyric acid.

[0008] To solve Fg To address the problem of poor DAAO activity, the present invention uses the Evolve pro model to predict single-point mutations with activity as the evaluation indicator. After only two rounds of single-point mutation iterations, the highly active Y242V single-point mutation was obtained. Compared with the wild type, its enzyme activity was significantly improved. For D,L-norvaline, its relative activity was increased by 8.4 times, and its half-life at 60°C (4.32 min) was 0.9 times that of the wild type (4.9 min); for D,L-2-aminobutyric acid, its relative activity was increased by 3.9 times, and its half-life at 60°C (4 min) was 0.7 times that of the wild type (5.4 min).

[0009] Finally, the two single-point mutations were combined to create the V53F / Y242V double-point mutation, which significantly improved both relative activity and stability against substrates. For D,L-norvaline, the relative activity increased by 7.9-fold, the half-life at 60°C increased by 11-fold, and the residual enzyme activity after 15 minutes at 60°C increased by 5.2-fold. For D,L-2-aminobutyric acid, the relative activity increased by 3.9-fold, the half-life at 60°C increased by 8.1-fold, and the residual enzyme activity after 15 minutes at 60°C increased by 7.2-fold.

[0010] The method of the present invention effectively improves Fg The soluble expression, stability and activity of DAAO are improved by solving the problem of the production rate of keto acids, such as 2-ketopentanoic acid and 2-ketobutyric acid; and further realizing the efficient and low-cost catalytic production of L-amino acids, such as L-norvaline and L-2-aminobutyric acid, by multi-enzyme cascade in industrialization.

[0011] The first aspect of the present invention protects a D-amino acid oxidase mutant, based on the amino acid sequence of the wild-type D-amino acid oxidase, the amino acid sequence of the mutant comprises substitutions of V53F and / or Y242V, and the amino acid sequence of the wild-type D-amino acid oxidase comprises the sequence shown in SEQ ID No. 1.

[0012] MANTIIVVGAGVSGLTSAYLLSKNKGNKITVVAKHMPGDYDIEYASPFAGANVCPMATQENSRWERRTWVEFKRLCEQVPEAGIHFQKCHIARRKKDVEEAKSSTFPDALFQEEPWYKELFEDFREQNPNEVTRGYDSGCEFTSVCINTAIYLPWLAGQCLKNGVVLKRTILTDISEAKKLS HTGKVPNIIVNATGLGSLKLGGVKDETMAPARGQIVVVRNESTPMLITSGVEDGGSDVMYLMQRAAGGGTILGGTYDVGNWESQPDPNIAQRIMQRIVEARPEVADGKGVKGLSIIRHAVGLRPWRKGGLRLEEEKLDDETWIVHNYGHSGWGYQGSYGCAEGVVELVDKVGKGAKAKL(SEQ IDNO.1) The base sequence encoding the amino acid sequence shown in SEQ ID NO.1 includes the sequence shown in SEQ ID NO.2.

[0013] In certain embodiments, the mutant comprises a V53F substitution. The V53F single-point mutation in the present invention significantly improves thermal stability. For the substrate D,L-norvaline, the residual enzyme activity after incubation at 60°C for 15 minutes reached 95%, a 6.3-fold increase compared to the wild-type residual enzyme activity of 15%. Furthermore, the half-life at 60°C increased from 4.9 minutes in the wild-type to 127 minutes, a 25-fold increase. For the substrate D,L-2-aminobutyric acid, the residual enzyme activity after incubation at 60°C for 15 minutes reached 99%, a 9.0-fold increase compared to the wild-type residual enzyme activity of 11%. Furthermore, the half-life at 60°C increased from 5.4 minutes in the wild-type to 189 minutes, a 35-fold increase. Furthermore, the relative activity of V53F against both substrates was also improved, with a 2.1-fold increase for D,L-norvaline and a 1.6-fold increase for D,L-2-aminobutyric acid.

[0014] In certain embodiments, the mutant comprises a substitution at Y242V. Compared to the wild-type, the Y242V single-point mutation of the present invention significantly improves enzyme activity, with relative activity towards D,L-norvaline increased by 8.4 times and relative activity towards D,L-2-aminobutyric acid increased by 3.9 times.

[0015] In certain embodiments, the mutant comprises substitutions of V53F and Y242V. The present invention discovered that the D-amino acid oxidase containing the double mutations exhibited a relative activity of 7.9 times that of the wild type (NVA) when catalyzing D,L-norvaline (D,L-NVA), a residual enzyme activity of approximately 5.2 times that of the wild type after incubation at 60°C for 15 minutes (NVA), and an half-life of 11 times that of the wild type. Furthermore, the D-amino acid oxidase containing the double mutations exhibited a relative activity of 3.69 times that of the wild type (ABA), a residual enzyme activity of approximately 7.2 times that of the wild type after incubation at 60°C for 15 minutes (ABA), and an half-life of 8.1 times that of the wild type when catalyzing D,L-2-aminobutyric acid (D,L-ABA).

[0016] In certain embodiments, the wild-type D-amino acid oxidase is derived from Fusarium graminearum ( Fusarium graminearum ).

[0017] Another aspect of the present invention protects an isolated polynucleotide encoding a mutant as described above.

[0018] Another aspect of the present invention protects a nucleic acid construct comprising the polynucleotide as described above.

[0019] Another aspect of the present invention protects a host cell comprising the nucleic acid construct as described above or having the polynucleotide as described above integrated into its genome.

[0020] In some embodiments, the host cell can be any cell that can be used for proteinase K recombinant production, for example, a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell can also be a eukaryotic organism, such as mammals, insects, plants or fungal cells. Such as the Escherichia coli of the present application.

[0021] Another aspect of the present invention protects a method for producing a D-amino acid oxidase mutant, comprising: (a) culturing the above host cell under conditions suitable for expressing the D-amino acid oxidase mutant; and (b) recovering the D-amino acid oxidase mutant.

[0022] Another aspect of the present invention provides a composition comprising a mutant as described above. The composition may comprise a D-amino acid oxidase mutant of the present invention as the primary enzyme component, e.g., a monocomponent composition. Alternatively, the composition may comprise multiple enzyme activities, e.g., one or more (e.g., several) enzymes selected from the group consisting of: catalase, glutamate dehydrogenase, formate dehydrogenase, protease, glucoamylase, β-amylase, pullulanase.

[0023] Another aspect of the present invention protects the use of the mutant as described above, or the polynucleotide as described above, or the nucleic acid construct as described above, or the host cell as described above, or the composition as described above in the preparation of keto acids or L-amino acids.

[0024] Another aspect of the present invention protects a method for preparing keto acid, which uses the mutant as described above, the polynucleotide as described above, the nucleic acid construct as described above, the host cell as described above, or the composition as described above to catalyze the substrate, wherein the substrate is selected from amino acids.

[0025] In some embodiments, the substrate is selected from one or more of D,L-norvaline (D,L-NVA) and D,L-2-aminobutyric acid (D,L-ABA).

[0026] In some embodiments, the catalytic temperature is 20-65°C, preferably 40°C.

[0027] Another aspect of the present invention protects a method for preparing L-amino acids, which uses the mutant described above, the polynucleotide described above, the nucleic acid construct described above, the host cell described above, or the composition described above to catalyze the reaction of a substrate.

[0028] In some embodiments, the catalysis further comprises adding one or more of glutamate dehydrogenase (GluDH), formate dehydrogenase (FDH) and catalase.

[0029] In some embodiments, the substrate is selected from one or more of D,L-norvaline (D,L-NVA) and D,L-2-aminobutyric acid (D,L-ABA).

[0030] In some embodiments, the catalyzed reaction system further comprises ammonium formate and NAD.

[0031] In certain embodiments, the catalytic system comprises 100-300 mM D, L-norvaline (D, L-NVA), 200-400 mM ammonium formate, 0.1-0.3 mM NAD + , 0.5-1.5g / L wet bacteria of D-amino acid oxidase mutant.

[0032] In certain embodiments, the amount of wet glutamate dehydrogenase added is 0.1-1.0 g / L, preferably 0.5 g / L.

[0033] In certain embodiments, the amount of wet cells of formate dehydrogenase added is 0.5-2.0 g / L, preferably 1 g / L.

[0034] In certain embodiments, the amount of wet catalase cells added is 3-7 g / L, preferably 5 g / L.

[0035] In some embodiments, the catalytic temperature is 20-65°C, preferably 40°C.

[0036] In some embodiments, the L-amino acid is selected from one or both of L-norvaline and L-2-aminobutyric acid.

[0037] Compared with the prior art, the present invention has the following beneficial effects: 1) The D-amino acid oxidase mutant of the present invention has high solubility expression, high stability, and high activity. Its half-life at 60°C can reach 35 times that of the wild-type amino acid oxidase, solving the problem of the production rate of 2-ketopentanoic acid and 2-ketobutyric acid.

[0038] 2) The D-amino acid oxidase mutant of the present invention can be used with other enzymes, such as glutamate dehydrogenase, formate dehydrogenase, and catalase, to achieve a multi-enzyme cascade in industrial production to efficiently and cost-effectively catalyze the production of L-amino acids, such as norvaline and L-2-aminobutyric acid, with a concentration of up to 199 mM and an ee value of 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the relative activity assay results of the top 30 single-point mutations with the WT and ESM predicted values ​​in Example 1.

[0040] Figure 2 The supernatant and precipitate gel images of the top 30 single-point mutants with WT and ESM predicted values ​​in Example 1 are shown.

[0041] (where labels 1, 2, 3, etc. correspond to Figure 2 (number before the mutant name in the Figure 3 The residual enzyme activity of the mutant in Example 1 after incubation at 60°C for 15 min Figure 4 These are the relative activity test results of the top 20 single-point mutations predicted by Evolvepro in the first round of Example 2 for D,L-norvaline and D,L-2-aminobutyric acid.

[0042] Figure 5 These are the relative activity test results of the second round of Evolvepro in Example 2 for the top 20 single-point mutations with predicted values ​​for D,L-norvaline and D,L-2-aminobutyric acid.

[0043] Figure 6 The relative activities of WT, V53F, Y242V, and V53F / Y242V against D,L-norvaline and D,L-2-aminobutyric acid in Example 3 and the residual enzyme activity determination results after incubation at 60°C for 15 min.

[0044] Figure 7 These are the half-life determination results for WT, V53F, Y242V, and V53F / Y242V for D,L-norvaline and D,L-2-aminobutyric acid in Example 3.

[0045] Figure 8 Schematic diagram of the process for preparing L-norvaline and L-2-aminobutyric acid by the four-enzyme cascade in Example 4.

[0046] Figure 9 This is a graph showing the concentration changes of substrates, intermediates, and final products over time during the four-enzyme cascade reaction to prepare L-norvaline and L-2-aminobutyric acid in Example 4. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. The following examples are only specific embodiments of the present invention, but protection scope of the present invention is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were performed using three replicates, and the results were averaged.

[0048] Reagents used in upstream genetic engineering: E. coli BL21 (DE3) was purchased from Novagen; plasmid pET-28a(+) and others were purchased from Novagen; Fg The gene synthesis, primer synthesis and sequence sequencing of DAAO were completed by Qingke Bioengineering Co., Ltd.

[0049] The reagents used in the catalytic reaction: D,L-norvaline (D,L-NVA), L-norvaline (L-NVA), 4-aminoantipyrine, and horseradish peroxidase were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; D,L-2-aminobutyric acid (D,L-ABA), L-2-aminobutyric acid (L-ABA), D-norvaline (D-NVA), and 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0050] The composition of LB medium was as follows: 10 g / L tryptone, 10 g / L sodium chloride, and 10 g / L yeast extract. Solid LB medium was supplemented with 15 g / L agar. Tryptone and yeast extract were purchased from Thermo Fisher Scientific, and sodium chloride and agar were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0051] Definition of enzyme activity unit (U): the amount of enzyme required to produce 1 μmol of 2-ketovalerate (2-ketobutyrate) per minute in the reaction system.

[0052] Example 1 Fg Screening of single-point mutants with improved solubility and stability of DAAO Since wild type Fg During protein expression, DAAO often misfolds and aggregates, precipitating to form inclusion bodies. To improve its soluble expression and stability, the ESM model was used to predict and score dominant mutation points. A more positive score indicates a more favorable single-point mutation. Based on the predicted results, single-point mutations were constructed, followed by protein expression and thermal stability and activity verification. This included the following: 1.1. ESM prediction of single-point mutations that improve solubility and stability The wild type FgThe amino acid sequence of DAAO protein was input into the ESM model. A total of 6 ESM models were used for prediction. The number of models with positive scores for each single point mutation was counted, with the highest being 6 positive values ​​and the lowest being 0 positive values. The models were sorted according to the number of positive values. If the number of positive values ​​was the same, the models were sorted according to the maximum value. Finally, the top 30 single point mutations Q127I, Q127L, Q127M, V53W, C54L, Q127V, T133P, I279A, G354L, F48W, C76A, A92F, A92Y, E122P, I279C, A282L, E322G , Y19L, V53Y, V53F, V70P, Q127F, R134P, D259Q, R283C, V286L, K293E, A92H, A92W, P107F.

[0053] 1.2 Construction of single-point mutations (1) Whole plasmid PCR: Single point mutations were constructed using whole plasmid PCR, using pET28a- Fg Using the DAAO plasmid as a template, upstream and downstream primers covering the 30 single-point mutations in step 1.1 were designed (Table 1) and whole-plasmid PCR was performed.

[0054] Table 1 Primers used for the construction of DAAO mutants

[0055] The PCR amplification system was prepared as follows, and PCR amplification was performed.

[0056] PCR amplification system:

[0057] PCR amplification conditions: 1) Pre-denaturation: 98°C for 3 min; 2) Denaturation: 98°C for 10 s; Annealing: 60°C for 15 s; Extension: 72°C for 1 min; 33 cycles in total; 3) Post-extension: 72°C for 5 min; 4) Store at 4°C.

[0058] (2) Transformation and verification: The PCR product was transformed into E. coli BL21 (DE3) competent cells using the heat shock method. Competent cells were removed from -80°C and thawed on ice for 2 minutes. 10 μL of the ligation product was mixed with 100 μL of E.coli BL21(DE3) competent cells and incubated on ice for 30 minutes. The centrifuge tube was heat-shocked in a 42°C water bath for 1 minute, immediately removed, and incubated on ice for 3 minutes. In a sterile operating hood, 600 μL of LB medium was added to the centrifuge tube, mixed thoroughly, and incubated at 200 rpm at 37°C for 60 minutes to revive the cells. After revival, the cells were centrifuged at 6000 rpm at room temperature for 30 seconds. The supernatant was partially discarded in a laminar flow hood, and the remaining liquid (approximately 100 μL) was mixed with the cells. The mixture was evenly spread on LB solid medium containing Kan antibiotics using a disposable spreader. Once the liquid was completely absorbed, the plate was inverted and placed in a 37°C incubator for approximately 16 hours. A single colony was selected from each plate for sequencing verification.

[0059] 1.3. Strain culture induction and cell disruption 1) Inoculate the recombinant E. coli obtained in step 1.2 into LB medium containing 50 μg / mL kanamycin and culture at 37°C, 200 rpm for 12 h to obtain seed solution.

[0060] 2) Inoculate the seed solution into fresh LB medium at a volume ratio of 2% and culture at 37°C, 200 rpm until the OD value of the bacterial solution reaches 600 When the p-value reached 0.8, IPTG solution was added to a final concentration of 0.5 mM, and the cells were induced in a shaking incubator at 18 °C and 200 rpm for 16 h.

[0061] 3) After induction culture, centrifuge at 4000 rpm for 15 min, discard the supernatant, and wash the bacterial pellet twice with phosphate buffer.

[0062] 4) The collected bacterial pellet was then resuspended in phosphate buffer and disrupted by ultrasonication at 400 W in an ice-water bath for 3 seconds each time with 7 seconds intervals until clear. The pellet was centrifuged at 8000 rpm for 10 minutes and the supernatant was used as the DAAO enzyme solution.

[0063] 1.4. DAAO activity, solubility and stability testing The amount of the byproduct H2O2 generated by the reaction of DAAO and substrate in the reaction system was quantitatively analyzed using a microplate reader to detect the enzyme activity.

[0064] Enzyme activity is measured using the Trinder reaction, which works as follows: In the presence of horseradish peroxidase, H₂O₂ oxidizes 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid to form a red quinoneimine. The absorbance of this red quinoneimine is measured at 510 nm using a microplate reader. The absorbance is proportional to the amount of quinoneimine generated, and the amino acid oxidase activity can be calculated. The details are as follows: 1) Prepare the colorimetric reagent for enzyme activity assay: Weigh 16 mg of 2,4,6-tribromo-3-hydroxybenzoic acid, 100 mg of 4-aminoantipyrine, and 20 mg of horseradish peroxidase, dissolve in 80 mL of pH 8 phosphate buffer, adjust the pH to 8, and finally dilute to 100 mL. Store in a refrigerator at 4°C, protected from light, to obtain the colorimetric reagent solution.

[0065] 2) Place the developer solution and 200 mM D,L-amino acid solution (D,L-NVA or D,L-ABA) in a thermostatic metal bath at 40°C for 10 min.

[0066] 3) Add 20 μL of the DAAO enzyme solution obtained in step 1.3, 100 μL of the colorimetric reagent solution, and 100 μL of the D,L-amino acid solution to the wells of the ELISA plate. Incubate at 40°C and detect using a microplate reader at a scanning wavelength of 510 nm, a scanning time of 180 s, and a scanning interval of 15 s.

[0067] 4) After the time scan is completed, the file is exported in Excel format and the data is processed. A graph is drawn with time as the horizontal axis (unit: min) and absorbance as the vertical axis. The slope value is obtained by linear fitting, and the enzyme activity is calculated based on this slope value.

[0068] Enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of ketoacid per minute under standard reaction conditions. Relative activity is the ratio of the mutant activity to the wild-type D-amino acid oxidase activity.

[0069] The relative activity test results of the top 30 single point mutations are shown in Figure 1 ; Protein gel images of supernatant and precipitate are shown in Figure 2 Among them, S stands for supernatant; p stands for precipitation; WT stands for wild type.

[0070] from Figure 1It can be seen that when D,L-NVA is used as the substrate, the single point mutations whose enzyme activity ratios relative to the wild type are greater than 1 include F48W, V53W, C54L and V53F; when D,L-ABA is used as the substrate, the single point mutations whose enzyme activity ratios relative to the wild type are greater than 1 include F48W, A92Y, Q127M, Q127V, I279A, A282L, Y19L, V53F, R134P, R283C and V286L.

[0071] The solubility of DAAO enzyme was characterized by SDS-PAGE protein electrophoresis. 18 μL of the supernatant and protein sample (obtained after cell disruption and centrifugation) were added to 6 μL of 4× Protein SDS PAGE Loading Buffer. Mix well, heat at 99°C for 10 minutes in a PCR instrument to fully denature the protein sample, and then cool to room temperature. A pre-assembled protein gel with a 4%–20% concentration was removed from the bottom seal and loaded into an electrophoresis tank. The electrophoresis buffer was then added, and the comb was removed to prepare the sample for loading. 10 μL of protein sample and protein marker were added to each well of the gel. The gel was powered on and protein electrophoresis was performed at 120 V. When the bromophenol blue indicator had just run off the bottom of the gel, the power was turned off. The gel was removed from the outer cover and placed in a staining vat containing staining solution. Heat for 30 seconds and stain on a reciprocating shaker for 30 minutes. The gel was then transferred to a destaining solution and destained repeatedly until the background color was completely removed.

[0072] from Figure 2 As can be seen, the relative ratios of the supernatant enzyme and precipitate for mutants numbered 1 (F48W), 6 (A92Y), 11 (I279A), and 20 (V53F) are higher than those for the wild type, indicating better solubility. A darker color in the supernatant relative to the precipitate indicates a higher concentration of soluble protein than precipitated protein, indicating better solubility.

[0073] Finally, the mutation points F48W, V53F, A92Y, and I279A that showed significant improvements in soluble expression and enzyme activity compared to the wild type were selected and thermal stability tests were performed. The results are as follows: Figure 3 shown.

[0074] Incubate the DAAO enzyme solution obtained in step 1.3 in a PCR instrument at 60°C for 15 min. Then, add 20 μL of the DAAO enzyme solution, 100 μL of the developer solution, and 100 μL of the D,L-amino acid solution to the wells of the microplate. Measure the enzyme activity after heat treatment using a microplate reader at 510 nm at 40°C. Calculate the residual enzyme activity. Residual enzyme activity = enzyme activity after heat treatment / enzyme activity before treatment.

[0075] from Figure 3It can be seen that among the four single-point mutations, the soluble expression of the V53F single-point mutation was improved, and the thermal stability was significantly improved compared with the wild type. For the substrate D,L-norvaline, the residual enzyme activity was still about 95% after being incubated at 60°C for 15 minutes, which was 6.3 times higher than the residual enzyme activity of 15% of the wild type; for the substrate D,L-2-aminobutyric acid, the residual enzyme activity reached 99% after being incubated at 60°C for 15 minutes, which was 9.0 times higher than the residual enzyme activity of 11% of the wild type.

[0076] Example 2 Fg Screening of DAAO activity-enhancing single-point mutants The Evolve pro model (Kaiyi Jiang et al. Rapid in silico directed evolution by a protein language model with EVOLVEpro. Science, 2024) was used to predict single-point mutations using activity as the evaluation indicator.

[0077] The activity data of the first 30 single-point mutations predicted by ESM were used as the initial input data set. In each round, the top 20 single-point mutations with predicted output values ​​for the two substrates (40 single-point mutations in total) were selected for activity detection experiments. The experimental results were then input into the Evolve pro model of the corresponding substrates. A total of 2 rounds of single-point mutation iterations were performed. The results of the first round of single-point mutation iterations are shown in Figure 4 The results of the second round of single point mutation iterations are shown in Figure 5 .

[0078] Figure 4 These are the results of the first round of Evolvepro relative activity assays for the top 20 single-point mutations predicted for D,L-norvaline and D,L-2-aminobutyric acid.

[0079] from Figure 4It can be seen that when D,L-NVA is used as the substrate, the single point mutations with enzyme activity greater than 1 relative to the wild type are F48N, K309S, I229W, F48M, K186D, F48M, K186E, K180T, K180W, L200T, K290M, K102G, R64E, F106C, L200Q, L200A, and K102D; when D,L-ABA is used as the substrate, the enzyme activity relative to the wild type is greater than 1. The single point mutations with ratios greater than 1 are I229A, I229T, I229S, F48N, K309S, I229W, F48M, I229C, K180M, K180E, K180D, I279V, K186D, K180V, K290E, F48S, K186E, I325V, K180T, I151T, N270G, K180W, L200T, and K290M.

[0080] Figure 5 These are the relative activity results of the second round of Evolvepro targeting the top 20 single-point mutations with the predicted values ​​for D,L-norvaline and D,L-2-aminobutyric acid.

[0081] from Figure 5 It can be seen that when D,L-NVA is used as the substrate, the single point mutations whose enzyme activity ratios relative to the wild type are greater than 1 include K309E, K186T, K180H, K180Q, K358E, I229D, Y242V, I229K, I229N, Y242L, and L110G; when D,L-ABA is used as the substrate, the single point mutations whose enzyme activity ratios relative to the wild type are greater than 1 include Y242V, Y242L, K180S, K309E, K186T, K180H, K180Q, K358E, and K23T.

[0082] Table 2 Primers used for the construction of DAAO mutants

[0083] In summary, the single point mutation Y242V has the highest activity improvement against both substrates.

[0084] Example 3 Fg Construction of DAAO double-point mutations The single point mutation V53F with the best solubility and stability predicted by the ESM model and Y242V with the most significant activity improvement predicted by the Evolve pro model were combined to obtain the double point mutation V53F / Y242V.

[0085] The construction method remained the same as that of whole-plasmid PCR in Example 1. First, a plasmid with the Y242V mutation was extracted from the successfully constructed recombinant bacteria. Whole-plasmid PCR was then performed on the Y242V sequence using primers targeting V53F to generate the double-site combination V53F / Y242V. The enzyme was then transformed into competent E. coli cells, induced with IPTG, and then centrifuged and sonicated to obtain a crude enzyme solution. This was done in the same manner as steps 1.2 and 1.3 of Example 1.

[0086] The relative activity, residual enzyme activity after incubation at 60°C for 15 minutes, and half-life t1 / 2 at 60°C were measured for WT, V53F, Y242V, and V53F / Y242V. The half-life was determined by incubating the crude enzyme solution at 60°C, measuring activity at regular intervals, and calculating the residual enzyme activity. The incubation time at which the residual activity reached 50% was the half-life of the enzyme at 60°C. Figure 6 and Figure 7 shown.

[0087] from Figure 6 It can be seen that for D,L-norvaline, compared with the wild type, the relative activity of the Y242V single point mutation was significantly increased by 8.4 times; compared with the wild type, the relative activity of the V53F single point mutation was also improved to a certain extent, and its relative activity increased by 2.1 times; compared with the wild type, the double point mutation V53F / Y242V had significantly improved thermal stability and relative activity compared with the wild type, with a relative activity of 7.9 times that of the wild type (NVA), and the residual enzyme activity after incubation at 60°C for 15 min was approximately 5.2 times that of the wild type (NVA).

[0088] from Figure 6 It can be seen that for D,L-2-aminobutyric acid, compared with the wild type, the relative activity of the Y242V single point mutation increased by 3.9 times; compared with the wild type, the relative activity of the V53F single point mutation increased by 1.6 times; compared with the wild type, the double point mutation V53F / Y242V had significantly improved thermal stability and activity compared with the wild type, with a relative activity of 3.69 times that of the wild type (ABA), and the residual enzyme activity after incubation at 60°C for 15 min was approximately 7.2 times that of the wild type (ABA).

[0089] from Figure 7 It can be seen that when D,L-norvaline (D,L-NVA) is used as the substrate, the 60℃ half-life of the V53F single mutant (127 min) is 25 times that of the wild type (4.9 min); the 60℃ half-life of Y242V (4.32 min) is 0.9 times that of the wild type (4.9 min); and the 60℃ half-life of the double-point mutation V53F / Y242V (53.9 min) is 11 times that of the wild type (4.9 min).

[0090] from Figure 7It can be seen that when D,L-2-aminobutyric acid (D,L-ABA) is used as the substrate, the 60℃ half-life of the V53F single mutant (189 min) is 35 times that of the wild type (5.4 min); the 60℃ half-life of Y242V (4 min) is 0.7 times that of the wild type (5.4 min); and the 60℃ half-life of the double-point mutation V53F / Y242V (43.74 min) is 8.1 times that of the wild type (5.4 min).

[0091] Example 4: Cascade preparation of L-short-chain unnatural amino acids using D-amino acid oxidase, catalase, glutamate dehydrogenase, and formate dehydrogenase The schematic diagram of the four-enzyme cascade for the preparation of L-norvaline (L-2-aminobutyric acid) is shown in Figure 8 Glutamate dehydrogenase is derived from Clostridium difficile 630, is a wild-type NAD(H)-dependent GluDH mutated to contain a single point mutation K71A (NCBI reference sequence: WP_003420866.1), the amino acid sequence of the GluDH containing the single point mutation K71A is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4. Formate dehydrogenase is derived from Starkeya nomas The wild type (NCBI reference sequence: WP_159599443.1), its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6.

[0092] According to the induction expression and cell disruption method described in Example 1, the cells were transferred into E. coli BL21 D-amino acid oxidase, glutamate dehydrogenase, and formate dehydrogenase of (DE3) were induced to disrupt the cells and obtain the supernatant enzyme solution. Glutamate dehydrogenase and formate dehydrogenase were also expressed in the pET28a vector using the same induction expression method as D-amino acid oxidase.

[0093] The catalytic system for the D,L-norvaline (D,L-NVA) substrate was as follows: enzyme solution was added to a 200 mL reaction system in the form of the supernatant. The initial addition amounts of the four enzymes were 1 g / L wet weight of bacterial DAAO-V53F / Y242V, 0.5 g / L wet weight of bacterial GluDH, 1 g / L wet weight of bacterial FDH, and 5 μL / L commercial catalase (CAT); 200 mM D,L-norvaline (D,L-NVA), 300 mM ammonium formate, 0.2 mM NAD+, pH 8.0, and a temperature of 40°C. The reaction was carried out under aerobic conditions for 6 h, and samples were taken every 1 h to detect the substrate and product.

[0094] The catalytic system for the D,L-ABA substrate is as follows: The difference from the catalytic system for the D,L-norvaline (D,L-NVA) substrate is that D,L-ABA is used instead of D,L-NVA, and everything else is the same.

[0095] Liquid chromatography detection conditions for intermediate products: chromatographic column model / Pntulips® QS-C18, 5μm, 4.6mm×250mm; detection wavelength / UV 205 nm; column temperature / 40℃; injection volume / 20 μL; flow rate / 1 mL / min; mobile phase / 50 mM (NH4)2HPO4 aqueous solution:acetonitrile = 95:5.

[0096] Substrates and end products were determined by pre-column liquid chromatography derivatization. Derivatization reagent preparation: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine, add 400 μL of anhydrous ethanol and 4 mL of boric acid buffer, and sonicate to completely dissolve. Prepare freshly prepared samples for immediate use. Derivatization reaction and determination: Add 100 μL of the sample to 100 μL of the derivatization reagent, mix thoroughly, and incubate at 25°C for 5 minutes. Chromatographic conditions: Column model: Pintulips® QS-C18, 5 μm, 4.6 mm × 250 mm; detection wavelength: 334 nm; column temperature: 30°C; injection volume: 20 μL; flow rate: 1 mL / min; mobile phase: 50 mM sodium acetate in water: methanol (50:50).

[0097] Figure 9 This graph shows the time-dependent concentration changes of substrates, intermediates, and final products during the four-enzyme cascade reaction to produce L-norvaline and L-2-aminobutyric acid. The substrates are D,L-norvaline (D-NVA) and D,L-2-aminobutyric acid (D-ABA); the intermediates are 2-ketovalerate and 2-ketobutyrate; and the final products are L-norvaline (L-NVA) and L-2-aminobutyric acid (L-ABA).

[0098] from Figure 9 It can be seen from the HPLC determination results that the concentration of the final products L-norvaline and L-2-aminobutyric acid can reach 199mM, and the ee value can reach 99%.

[0099] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. A D-amino acid oxidase mutant, characterized in that Based on the amino acid sequence of the wild-type D-amino acid oxidase, the amino acid sequence of the mutant comprises substitutions of V53F and / or Y242V, and the amino acid sequence of the wild-type D-amino acid oxidase comprises the sequence shown in SEQ ID No.

1.

2. The mutant according to claim 1, characterized in that The mutant contains substitutions of V53F and Y242V.

3. An isolated polynucleotide, characterized in that Encoding the mutant according to claim 1 or 2.

4. A nucleic acid construct, characterized in that Comprising the polynucleotide according to claim 3.

5. A host cell, characterized in that The nucleic acid construct according to claim 4 is included, or the polynucleotide according to claim 3 is integrated into the genome.

6. A composition, characterized in that Comprising the mutant according to claim 1 or 2.

7. The composition according to claim 6, wherein Also contains catalase, glutamate dehydrogenase and formate dehydrogenase.

8. Use of the mutant according to claim 1, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the host cell according to claim 5, or the composition according to claim 6 in the preparation of keto acids or L-amino acids.

9. A method for preparing keto acid, characterized in that: The mutant according to claim 1, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the host cell according to claim 5, or the composition according to claim 6 is used to catalyze the substrate, wherein the substrate is selected from amino acids.

10. A method for preparing L-amino acids, characterized in that: The substrate is catalyzed using the mutant according to claim 1, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the host cell according to claim 5, or the composition according to claim 6.

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