A D-amino acid oxidase mutant and its application

Abstract

This invention discloses a D-amino acid oxidase mutant and its applications. Based on the amino acid sequence of wild-type D-amino acid oxidase, the mutant's amino acid sequence includes substitutions of V53F and / or Y242V, while the wild-type D-amino acid oxidase's amino acid sequence includes the sequence shown in SEQ ID No. 1. The D-amino acid oxidase mutant of this invention exhibits high catalytic activity and high stability.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a D-amino acid oxidase mutant and its applications. Background Technology

[0002] Short-chain non-natural amino acids have wide applications in medicine, chemical industry, and agriculture due to their structural diversity and rich functionality. However, their synthesis is relatively difficult, and the modification of key enzymes and the development of catalytic processes in their synthesis have significant theoretical and potential economic value. The target products of this invention are L-valine and L-2-aminobutyric acid (GABA). L-valine is used as a key intermediate in the synthesis of perindopril, a common antihypertensive drug, while L-2-aminobutyric acid is commonly used in the synthesis of ethambutol, an anti-tuberculosis drug, and levetiracetam and brivaracetam, antiepileptic drugs. Methods for synthesizing these short-chain non-natural amino acids include chemical methods and biocatalytic methods. Chemical methods typically suffer from low optical purity, harsh reaction conditions, and cumbersome procedures. Compared to chemical synthesis methods, biocatalytic methods can economically, greenly, and efficiently synthesize L-valine and L-2-aminobutyric acid with high chiral purity, demonstrating 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 flavin protease class. Under aerobic conditions, it selectively catalyzes the oxidative dehydrogenation of D-amino acids to form the corresponding α-imino acids. The produced imino acids can spontaneously hydrolyze to α-keto acids and ammonia. Simultaneously, oxygen molecules re-oxidize the reduced FAD to generate H2O2. D-amino acid oxidase can be used in a four-enzyme one-pot process with catalase, amino acid dehydrogenase, and formate dehydrogenase to produce L-short-chain non-natural amino acids. The main reaction process involves the first oxidation of D-type amino acids in a mixed substrate to keto acids by DAAO in the system. Catalase removes the byproduct H2O2, followed by a cascade reaction with amino acid dehydrogenase. Formate dehydrogenase then performs a coenzyme cycle to convert the keto acid into L-short-chain non-natural amino acids, which are then racemiced to produce L-short-chain non-natural amino acids.

[0004] Currently reported D-amino acid oxidases generally suffer from low activity, moderate thermostability, and poor soluble expression in E. coli expression systems for substrates D-valine and D-2-aminobutyric acid. 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 target products. Summary of the Invention

[0005] To address the problems of low catalytic activity, poor thermal stability, and poor soluble expression of D-amino acid oxidases in the Escherichia coli expression system in the prior art, this invention provides a D-amino acid oxidase mutant and its application.

[0006] This invention first screened the DAAO enzyme library for enzymes derived from Fusarium graminearum that exhibited high activity against the substrates D-valine and D-2-aminobutyric acid. Fusarium graminearum D-amino acid oxidase Fg DAAO. Then from Fg Starting with DAAO, the gene was modified through site-directed mutagenesis to improve... Fg The catalytic activity and stability of DAAO were studied to achieve its soluble expression in Escherichia coli, thereby further improving the production rate of 2-pentanone and 2-butanone, and laying the foundation for more efficient industrial production of L-short-chain non-natural amino acids through multi-enzyme cascades.

[0007] To solve Fg To address the issues of DAAO's soluble expression and poor stability, this invention first utilizes the ESM model to... Fg Single-point mutations predicted by DAAO sequence analysis yielded the V53F mutation. This mutation significantly improved thermostability. For the substrate D,L-valine, the residual enzyme activity reached 95% after 15 minutes of incubation at 60°C, a 6.3-fold increase compared to the wild-type residual activity of 15%. Furthermore, the half-life at 60°C increased from 4.9 min in the wild-type to 127 min, a 25-fold increase. For the substrate D,L-2-aminobutyric acid (GABA), the residual enzyme activity reached 99% after 15 minutes of incubation at 60°C, a 9.0-fold increase compared to the wild-type residual activity of 11%. The half-life at 60°C also increased from 5.4 min in the wild-type to 189 min, a 35-fold increase. Simultaneously, the relative activity of V53F against both substrates was also improved: a 2.1-fold increase in relative activity against D,L-valine and a 1.6-fold increase in relative activity against D,L-2-aminobutyric acid.

[0008] To solve Fg To address the issue of poor DAAO activity, this invention uses the Evolve pro model to predict single-point mutations with activity as the evaluation index. A highly active Y242V single-point mutation was obtained through only two rounds of iteration. Compared to the wild type, its enzyme activity is significantly improved. For D,L-valine, its relative activity is increased by 8.4 times, and its half-life at 60℃ (4.32 min) is 0.9 times that of the wild type (4.9 min). For D,L-2-aminobutyric acid, its relative activity is increased by 3.9 times, and its half-life at 60℃ (4 min) is 0.7 times that of the wild type (5.4 min).

[0009] Finally, the two single-point mutations were combined to obtain the V53F / Y242V double-point mutation, which significantly improved the relative activity and stability of the substrate. For D,L-valine, the relative activity increased by 7.9 times, the half-life at 60℃ increased by 11 times, and the residual enzyme activity after 15 min of incubation at 60℃ increased by 5.2 times; for D,L-2-aminobutyric acid, the relative activity increased by 3.9 times, the half-life at 60℃ increased by 8.1 times, and the residual enzyme activity after 15 min of incubation at 60℃ increased by 7.2 times.

[0010] The method of the present invention effectively improves Fg The soluble expression, stability, and activity of DAAO can be improved by addressing the production rate issues of keto acids, such as 2-pentanone and 2-butanone; and further, the efficient and low-cost catalytic production of L-amino acids, such as L-valine and L-2-aminobutyric acid, can be achieved in industrial applications.

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

[0012] MANTIIVVGAGVSGLTSAYLLSKNKGNKITVVAKHMPGDYDIEYASPFAGANVCPMATQENSRWERRTWVEFKRLCEQVPEAGIHFQKCHIARRKKDVEEAKSSTFPDALFQEEPWYKELFEDFREQNPNEVTRGYDSGCEFTSVCINTAIYLPWLAGQCLKNGVVLKRTILTDISEAKKLS HTGKVPNIIVNATGLGSLKLGGVKDETMAPARGQIVVVRNESTPMLITSGVEDGGSDVMYLMQRAAGGGTILGGTYDVGNWESQPDPNIAQRIMQRIVEARPEVADGKGVKGLSIIRHAVGLRPWRKGGLRLEEEKLDDETWIVHNYGHSGWGYQGSYGCAEGVVELVDKVGKGAKAKL(SEQ IDNO.1)

[0013] The base sequence encoding the amino acid sequence shown in SEQ ID NO.1 includes the sequence shown in SEQ ID NO.2.

[0014] In some embodiments, the mutant comprises a substitution of V53F. The single-point mutation of V53F in this invention significantly improves thermal stability. For the substrate D,L-valine, the residual enzyme activity after incubation at 60°C for 15 minutes reaches 95%, a 6.3-fold increase compared to the wild-type residual enzyme activity of 15%, and the half-life at 60°C increases from 4.9 min for the wild-type to 127 min, a 25-fold increase. For the substrate D,L-2-aminobutyric acid (GABA), the residual enzyme activity after incubation at 60°C for 15 minutes reaches 99%, a 9.0-fold increase compared to the wild-type residual enzyme activity of 11%, and the half-life at 60°C increases from 5.4 min for the wild-type to 189 min, a 35-fold increase. Simultaneously, the relative activity of V53F for both substrates is also improved: the relative activity for D,L-valine is increased by 2.1 times; and the relative activity for D,L-2-aminobutyric acid is increased by 1.6 times.

[0015] In some embodiments, the mutant comprises a substitution of Y242V. Compared to the wild type, the enzyme activity of the Y242V single-point mutation in this invention is significantly increased, with a relative increase of 8.4 times in activity against D,L-valine and a relative increase of 3.9 times in activity against D,L-2-aminobutyric acid.

[0016] In some embodiments, the mutant comprises substitutions for V53F and Y242V. This invention has found that the D-amino acid oxidase containing the double mutation point exhibits a relative activity 7.9 times (NVA) higher than the wild type when catalyzing D,L-valine (D,L-NVA), with a residual enzyme activity approximately 5.2 times (NVA) higher after 15 min incubation at 60°C, and a half-life 11 times longer than the wild type at 60°C; when catalyzing D,L-2-aminobutyric acid (D,L-ABA), the relative activity is 3.69 times (ABA) higher than the wild type, with a residual enzyme activity approximately 7.2 times (ABA) higher after 15 min incubation at 60°C, and a half-life 8.1 times longer than the wild type at 60°C.

[0017] In some embodiments, the wild-type D-amino acid oxidase is derived from Fusarium graminearum (…). Fusarium graminearum ).

[0018] Another aspect of the present invention protects an isolated polynucleotide encoding the mutant described above.

[0019] Another aspect of the present invention protects a nucleic acid construct comprising the polynucleotides described above.

[0020] Another aspect of the present invention protects a host cell comprising a nucleic acid construct as described above or a genome in which polynucleotides as described above are integrated.

[0021] In some embodiments, the host cell can be any cell capable of being used for proteinase K recombinant production, such as prokaryotic or eukaryotic cells. 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*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. Gram-negative bacteria include, but are not limited to, *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell, such as *Escherichia coli* as described in this application.

[0022] Another aspect of the present invention protects a method for producing a D-amino acid oxidase mutant, comprising: (a) culturing the aforementioned host cells under conditions suitable for expressing the D-amino acid oxidase mutant; and (b) recovering the D-amino acid oxidase mutant.

[0023] Another aspect of the present invention protects a composition comprising the mutant described above. The composition may comprise the D-amino acid oxidase mutant of the present invention as the main enzyme component, for example, a single-component composition. Alternatively, the composition may comprise a variety of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: catalase, glutamate dehydrogenase and formate dehydrogenase, protease, glucosylamylase, β-amylase, and amylopectinase.

[0024] Another aspect of the present invention protects the use of the mutants, polynucleotides, nucleic acid constructs, host cells, or compositions described above in the preparation of keto acids or L-amino acids.

[0025] Another aspect of the present invention protects a method for preparing keto acids, which uses a mutant, a polynucleotide, a nucleic acid construct, a host cell, or a composition as described above to catalyze a substrate selected from amino acids.

[0026] In some embodiments, the substrate is selected from one or more of D,L-n-valine (D,L-NVA) and D,L-2-aminobutyric acid (D,L-ABA).

[0027] In some embodiments, the catalytic temperature is 20-65°C. Preferably, it is 40°C.

[0028] Another aspect of the present invention protects a method for preparing L-amino acids, which uses a mutant, a polynucleotide, a nucleic acid construct, a host cell, or a composition as described above to catalyze a substrate.

[0029] In some embodiments, catalysis also includes the addition of one or more of glutamate dehydrogenase (GluDH), formate dehydrogenase (FDH), and catalase.

[0030] In some embodiments, the substrate is selected from one or more of D,L-n-valine (D,L-NVA) and D,L-2-aminobutyric acid (D,L-ABA).

[0031] In some embodiments, the catalytic reaction system also includes ammonium formate and NAD.

[0032] In some embodiments, the catalytic system comprises, based on the total volume of the reaction system, 100-300 mM D,L-n-valine (D,L-NVA), 200-400 mM ammonium formate, and 0.1-0.3 mM NAD. + 0.5-1.5 g / L wet bacterial cells of D-amino acid oxidase mutant.

[0033] In some embodiments, the amount of wet bacterial cells of the glutamate dehydrogenase added is 0.1-1.0 g / L. Preferably, it is 0.5 g / L.

[0034] In some embodiments, the amount of wet cells of the formate dehydrogenase added is 0.5-2.0 g / L. Preferably, it is 1 g / L.

[0035] In some embodiments, the amount of wet cells of catalase added is 3-7 g / L. Preferably, it is 5 g / L.

[0036] In some embodiments, the catalytic temperature is 20-65°C. Preferably, it is 40°C.

[0037] In some embodiments, the L-amino acid is selected from one or both of L-valine and L-2-aminobutyric acid.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1) The D-amino acid oxidase mutant of the present invention has high solubility, high stability and high activity. Its half-life at 60°C can reach 35 times that of wild-type amino acid oxidase, which solves the problem of the production rate of 2-pentanone and 2-butanone.

[0040] 2) The D-amino acid oxidase mutant of the present invention can be used in industrial production with other enzymes, such as glutamate dehydrogenase, formate dehydrogenase and catalase, to achieve efficient and low-cost catalytic production of L-amino acids, such as valine and L-2-aminobutyric acid, with a concentration of 199 mM and an ee value of 99%. Attached Figure Description

[0041] Figure 1 The graph shows the relative activity determination results of the top 30 single-point mutations in WT and ESM predicted values ​​in Example 1.

[0042] Figure 2 The images show the supernatant and precipitate gel images of the top 30 single-point mutations in WT and ESM predicted values ​​in Example 1.

[0043] (where numbers 1, 2, 3, etc. correspond to) Figure 2 (Prefix of the mutant name in the text)

[0044] Figure 3 The residual enzyme activity results of the mutant in Example 1 after incubation at 60 °C for 15 min.

[0045] Figure 4 The results of the first round of Evolvepro single-point mutation relative activity assays for the top 20 predicted values ​​of D,L-valine and D,L-2-aminobutyric acid in Example 2 are shown.

[0046] Figure 5 The results of the second round of Evolvepro single-point mutation relative activity assays for the top 20 predicted values ​​of D,L-valine and D,L-2-aminobutyric acid in Example 2 are as follows.

[0047] Figure 6 The results show the relative activities of WT, V53F, Y242V, and V53F / Y242V against D,L-valine and D,L-2-aminobutyric acid in Example 3, and the residual enzyme activity after incubation at 60 °C for 15 min.

[0048] Figure 7 The results of half-life determination for D,L-valine and D,L-2-aminobutyric acid using WT, V53F, Y242V, and V53F / Y242V in Example 3 are shown.

[0049] Figure 8 This is a schematic diagram of the process for preparing L-valine and L-2-aminobutyric acid using a four-enzyme cascade in Example 4.

[0050] Figure 9 The graph shows the changes in the concentrations of substrate, intermediate, and final products over time during the four-enzyme cascade preparation of L-valine and L-2-aminobutyric acid in Example 4. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments. The following examples are merely specific embodiments of the invention, but the scope of protection of the invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. In the quantitative experiments in the following embodiments, three replicate experiments were set up, and the results were averaged.

[0052] Reagents used in upstream genetic engineering: those used in the embodiments of this invention E. coli BL21 (DE3) was purchased from Novagen; plasmid pET-28a(+) and other components were purchased from Novagen. Fg The gene synthesis, primer synthesis, and sequence sequencing of DAAO were completed by Qingke Biotechnology Co., Ltd.

[0053] The reagents used in the catalytic reaction were: D,L-n-valine (D,L-NVA), L-n-valine (L-NVA), 4-aminoantipyridine, and horseradish peroxidase, which were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; D,L-2-aminobutyric acid (D,L-ABA), L-2-aminobutyric acid (L-ABA), D-n-valine (D-NVA), and 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA), which were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0054] The LB medium consisted of 10 g / L tryptone, 10 g / L sodium chloride, and 10 g / L yeast extract. Solid LB medium was supplemented with an additional 15 g / L agar. The tryptone and yeast extract were purchased from Thermo Fisher Scientific, while the sodium chloride and agar were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0055] The definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol of 2-pentanone (2-butanone) per minute in the reaction system.

[0056] Example 1 Fg DAAO improves solubility and stability, enhancing single-point mutant screening.

[0057] Due to wild type Fg DAAO protein expression frequently results in misfolding and aggregation, forming inclusion bodies. To improve its soluble expression and stability, an ESM model was used to predict and score dominant mutation sites; a higher score indicates a better single-point mutation. Based on the prediction results, single-point mutations were constructed, followed by protein expression and validation of thermostability and activity. This included the following:

[0058] 1.1 ESM predicts improved solubility and stability through single-point mutations

[0059] wild type Fg The amino acid sequence of the DAAO protein was input into an ESM model. Six ESM models were used for prediction. The number of models with positive scores for each single-point mutation was counted, with a maximum of 6 positive scores and a minimum of 0 positive scores. Models were ranked according to the number of positive scores; if the number of positive scores was the same, they were ranked according to the highest value. The top 30 single-point mutations were finally selected: 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.

[0060] 1.2 Construction of Single-Point Mutation

[0061] (1) Whole plasmid PCR: Single-point mutations were constructed using whole plasmid PCR, with pET28a- Fg Using the DAAO plasmid as a template, upstream and downstream primers (Table 1) covering the 30 single-point mutations in step 1.1 were designed for whole plasmid PCR.

[0062] Table 1 Primers used for constructing DAAO mutants

[0063]

[0064]

[0065] Prepare the PCR amplification system as follows and perform PCR amplification.

[0066] PCR amplification system:

[0067]

[0068] PCR amplification conditions:

[0069] 1) Pre-denaturation: 98℃ for 3 min; 2) Denaturation: 98℃ for 10 s; Annealing: 60℃ for 15 s; Extension: 72℃ for 1 min; 33 cycles in total; 3) Post-extension: 72℃ for 5 min; 4) Store at 4℃.

[0070] (2) Transformation and verification: The PCR product was transformed into E. coli BL21(DE3) competent cells by heat shock method. Competent cells were removed from -80°C and thawed on ice for 2 min. 10 μL of the ligation product was mixed with 100 μL of L / C coli BL21(DE3) competent cells and incubated on ice for 30 min. The centrifuge tube was then heat-shocked in a 42°C water bath for 1 min, immediately removed, and incubated on ice for 3 min. In a sterile environment, 600 μL of LB medium was added to the centrifuge tube, mixed well, and incubated at 37°C for 200 rpm for 60 min to revive the cells. After revival, the cells were centrifuged at 6000 rpm at room temperature for 30 s. Part of the supernatant was discarded in a laminar flow hood, and the remaining liquid (approximately 100 μL) was mixed with the cells. The mixture was then evenly spread onto LB solid medium containing Kans antibiotic using a disposable spreader. After complete absorption of the liquid, the plates were inverted and incubated at 37°C for approximately 16 h. Single colonies were picked from each plate for sequencing verification.

[0071] 1.3. Induction of bacterial strain culture and cell disruption

[0072] 1) The recombinant Escherichia coli obtained in step 1.2 was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37℃ and 200 rpm for 12 h to obtain seed culture.

[0073] 2) Inoculate the seed culture into fresh LB medium at a volume ratio of 2% and incubate at 37 ℃ and 200 rpm until the OD of the bacterial culture reaches a certain level. 600 Once the concentration reaches 0.8, add IPTG solution to a final concentration of 0.5 mM and induce for 16 h in a shaker at 18 ℃ and 200 rpm.

[0074] 3) After the induction culture is completed, centrifuge at 4000 rpm for 15 min, discard the supernatant, and wash the bacterial precipitate twice with phosphate buffer.

[0075] 4) The collected bacterial pellet was then resuspended in phosphate buffer and sonicated at 400 W in an ice-water bath for 3 seconds each time, with a 7-second interval, until clear. The pellet was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected as DAAO enzyme solution.

[0076] 1.4 Detection of DAAO activity, solubility and stability

[0077] The amount of H2O2, a byproduct generated from the reaction of DAAO and substrate in the reaction system, was quantitatively analyzed using an enzyme-linked immunosorbent assay (ELISA) reader to detect enzyme activity.

[0078] Enzyme activity was detected using the Trinder reaction. The principle is as follows: In the presence of horseradish peroxidase, H₂O₂ oxidizes 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid to generate a red quinone imine. The absorbance of this red quinone imine was measured at 510 nm using a microplate reader. The absorbance is directly proportional to the amount of quinone imine generated, and the enzyme activity of the amino acid oxidase can then be calculated. Details are as follows:

[0079] 1) Preparation of chromogenic 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, add 80 mL of phosphate buffer (pH=8) to dissolve, adjust the pH to 8, and finally bring the volume to 100 mL. Store in a refrigerator at 4 ℃, protected from light, to obtain the chromogenic reagent solution.

[0080] 2) Place the colorimetric reagent solution and 200 mM D,L-amino acid solution (D,L-NVA or D,L-ABA) in a constant temperature metal bath and keep them at 40 ℃ for 10 min.

[0081] 3) Add 20 μL of the DAAO enzyme solution obtained in step 1.3, 100 μL of the chromogenic reagent solution and 100 μL of D,L-amino acid solution to the wells of the ELISA plate, react at 40 ℃, and detect using an ELISA reader with a scanning wavelength of 510 nm and a scanning time of 180 s, scanning once every 15 s.

[0082] 4) After the time scan is completed, export the file in Excel format and process the data. Plot the data with time as the x-axis (unit: min) and absorbance as the y-axis. Obtain the slope value by linear fitting. Calculate the enzyme activity based on this slope value.

[0083] Enzyme activity unit (U) definition: The amount of enzyme required to produce 1 μmol of keto acid per minute under standard reaction conditions. Relative activity refers to the ratio of the enzyme activity of the mutant to that of the wild-type D-amino acid oxidase.

[0084] The results of the detection of the relative activities of the top 30 single-point mutations are shown in [link to data]. Figure 1 See the gel image of the supernatant and precipitate. Figure 2 Where S represents supernatant supernatant; p represents precipitation; and WT represents wild type.

[0085] from Figure 1It can be seen that, using D,L-NVA as a substrate, the single-point mutations with an enzyme activity ratio greater than 1 relative to the wild type are F48W, V53W, C54L, and V53F; using D,L-ABA as a substrate, the single-point mutations with an enzyme activity ratio greater than 1 relative to the wild type are F48W, A92Y, Q127M, Q127V, I279A, A282L, Y19L, V53F, R134P, R283C, and V286L.

[0086] The solubility of DAAO enzyme was characterized by SDS-PAGE protein electrophoresis. 18 μL of the supernatant enzyme solution and precipitated protein sample obtained after cell lysis and centrifugation were taken separately, and 6 μL of 4×Protein SDS-PAGE Loading Buffer was added. After mixing, the mixture was heated in a PCR instrument at 99℃ for 10 min to fully denature the protein sample, and then cooled to room temperature. Pre-prepared protein gels with a concentration of 4%–20% were used. After removing the bottom sealing film, the gels were placed in the electrophoresis tank, and electrophoresis buffer was poured in. The combs were removed, and the gels were ready for sample loading. 10 μL of protein sample and protein marker were added to each well. Electrophoresis was performed at 120 V. When the bromophenol blue indicator just emerged from the bottom of the gel, the power was turned off. The electrophoresis gel was removed, the outer shell was removed, and the gel was placed in a staining jar containing staining solution. The gel was heated for 30 s and stained on a reciprocating destaining shaker for 30 min. The gel was then transferred to destaining solution and destained repeatedly until the background color was completely removed.

[0087] from Figure 2 It can be seen that the relative content ratio of supernatant enzyme solution to precipitate of mutants labeled 1 (F48W), 6 (A92Y), 11 (I279A), and 20 (V53F) is higher than that of wild type, indicating better solubility. The darker the color of the supernatant compared to the precipitate, the more soluble protein there is than precipitated protein, indicating better solubility.

[0088] Finally, mutations F48W, V53F, A92Y, and I279A, which showed significant improvements in soluble expression and enzyme activity compared to the wild type, were selected, and their thermostability was tested. The results are as follows: Figure 3 As shown.

[0089] The DAAO enzyme solution obtained in step 1.3 was incubated in a PCR instrument at 60 ℃ for 15 min. Then, 20 μL of the DAAO enzyme solution, 100 μL of chromogenic reagent solution, and 100 μL of D,L-amino acid solution were added to the wells of an ELISA plate. The plate was then measured at 510 nm using an ELISA reader at 40 ℃. The enzyme activity after heat treatment was calculated, and the residual enzyme activity was also calculated. Residual enzyme activity = enzyme activity after heat treatment / enzyme activity before treatment.

[0090] from Figure 3It was found that among the four single-point mutations, the V53F single-point mutation showed improved soluble expression and significantly improved thermostability compared to the wild type. For the substrate D,L-valine, after incubation at 60℃ for 15 min, the residual enzyme activity was still about 95%, which was 6.3 times higher than the 15% residual enzyme activity of the wild type. For the substrate D,L-2-aminobutyric acid, the residual enzyme activity reached 99% after incubation at 60℃ for 15 min, which was 9.0 times higher than the 11% residual enzyme activity of the wild type.

[0091] Example 2 Fg DAAO activity enhancement single-point mutant screening

[0092] 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 with activity as the evaluation index.

[0093] Using the top 30 single-point mutation activity data predicted by ESM as the initial input dataset, in each round, the top 20 single-point mutations (40 in total) for the predicted output values ​​of the two substrates were selected for activity detection experiments. The experimental results were then input into the Evolve pro model of the corresponding substrates. A total of two rounds of single-point mutation iterations were performed. The results of the first round of single-point mutation iterations are shown below. Figure 4 The results of the second round of single-point mutation iterations are shown below. Figure 5 .

[0094] Figure 4 The results represent the relative activity assays of the top 20 single-point mutations predicted by Evolvepro in the first round for D,L-valine and D,L-2-aminobutyric acid.

[0095] from Figure 4It can be seen that, using D,L-NVA as a substrate, the single-point mutations with an enzyme activity ratio greater than 1 relative to the wild type include F48N, K309S, I229W, F48M, K186D, F48M, K186E, K180T, K180W, L200T, K290M, K102G, R64E, F106C, L200Q, L200A, and K102D; using D,L-ABA as a substrate, the enzyme activity ratio relative to the wild type... Single-point mutations with a ratio greater than 1 include 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.

[0096] Figure 5 The results represent the relative activity assays of the top 20 single-point mutations predicted by Evolvepro in the second round for D,L-valine and D,L-2-aminobutyric acid.

[0097] from Figure 5 It is known that, using D,L-NVA as a substrate, the single-point mutations with an enzyme activity ratio greater than 1 relative to the wild type include K309E, K186T, K180H, K180Q, K358E, I229D, Y242V, I229K, I229N, Y242L, and L110G; and using D,L-ABA as a substrate, the single-point mutations with an enzyme activity ratio greater than 1 relative to the wild type include Y242V, Y242L, K180S, K309E, K186T, K180H, K180Q, K358E, and K23T.

[0098] Table 2 Primers used for constructing DAAO mutants

[0099]

[0100]

[0101]

[0102] In summary, the single-point mutation Y242V showed the greatest increase in activity against both substrates.

[0103] Example 3 Fg Construction of DAAO two-point mutation

[0104] The single-point mutation V53F, which has the best solubility and stability as predicted by the ESM model, and Y242V, which has the most significant activity enhancement as predicted by the Evolve pro model, were combined to obtain the double-point mutation V53F / Y242V.

[0105] The construction method remains the same as the full-plasmid PCR in Example 1. First, the Y242V mutant plasmid is extracted from the successfully constructed recombinant bacteria. Then, full-plasmid PCR is performed on the Y242V sequence using primers of V53F to mutate and obtain the double-spot combination V53F / Y242V. This is transformed into competent E. coli cells, and IPTG-induced expression is performed. The cells are then centrifuged and sonicated to obtain a crude enzyme solution. Steps 1.2 and 1.3 in Example 1 are followed.

[0106] The relative activities, residual enzyme activity after incubation at 60 °C for 15 min, and half-life (t1 / 2) at 60 °C were measured for WT, V53F, Y242V, and V53F / Y242V enzymes. The half-life was determined by incubating the crude enzyme solution at 60 °C, measuring activity at regular intervals, calculating the residual enzyme activity, and defining the incubation time at which the residual activity reached 50% as the enzyme's half-life at 60 °C. Results are as follows: Figure 6 and Figure 7 As shown.

[0107] from Figure 6 It was found that, for D,L-valine, the relative activity of the Y242V single-point mutation was significantly increased by 8.4 times compared to the wild type; the relative activity of the V53F single-point mutation was also improved to some extent compared to the wild type, with a relative activity increase of 2.1 times; compared to the wild type, the thermostability and relative activity of the double-point mutant V53F / Y242V were significantly improved, with a relative activity of 7.9 times (NVA) of the wild type, and the residual enzyme activity after incubation at 60℃ for 15 min was approximately 5.2 times (NVA) of the wild type.

[0108] from Figure 6 It can be seen that, for D,L-2-aminobutyric acid, the relative activity of the Y242V single-point mutation is 3.9 times higher than that of the wild type; the relative activity of the V53F single-point mutation is 1.6 times higher than that of the wild type; and the thermostability and activity of the double-point mutant V53F / Y242V are significantly improved compared to the wild type, with a relative activity of 3.69 times (ABA) of the wild type, and the residual enzyme activity after incubation at 60℃ for 15 min is about 7.2 times (ABA) of the wild type.

[0109] from Figure 7 It can be seen that, when D,L-n-valine (D,L-NVA) is used as a substrate, the half-life of the V53F single mutant at 60℃ (127 min) is 25 times that of the wild type (4.9 min); the half-life of Y242V at 60℃ (4.32 min) is 0.9 times that of the wild type (4.9 min); and the half-life of the double-point mutant V53F / Y242V at 60℃ (53.9 min) is 11 times that of the wild type (4.9 min).

[0110] from Figure 7 It can be seen that, when D,L-2-aminobutyric acid (D,L-ABA) is used as a substrate, the half-life at 60℃ of the V53F single mutant (189 min) is 35 times that of the wild type (5.4 min); the half-life at 60℃ of Y242V (4 min) is 0.7 times that of the wild type (5.4 min); and the half-life at 60℃ of the double-point mutant V53F / Y242V (43.74 min) is 8.1 times that of the wild type (5.4 min).

[0111] Example 4: Preparation of L-short-chain non-natural amino acids by cascade of D-amino acid oxidase, catalase, glutamate dehydrogenase and formate dehydrogenase

[0112] A schematic diagram of the four-enzyme cascade preparation of L-valine (L-2-aminobutyric acid) is shown below. Figure 8 Glutamate dehydrogenase originates from Clostridium difficile 630 is an NAD(H)-dependent GluDH wild-type mutant containing 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) has the amino acid sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6.

[0113] According to the induction expression and cell disruption methods described in Example 1, the cells were transfected with... E. coli BL21 (DE3) D-amino acid oxidase, glutamate dehydrogenase, and formate dehydrogenase were induced to lyse cells and obtain supernatant enzyme solutions. Among them, glutamate dehydrogenase and formate dehydrogenase were also expressed using the pET28a vector, and their induction expression method was the same as that of D-amino acid oxidase.

[0114] The catalytic system for D,L-valine (D,L-NVA) substrate was as follows: Enzyme solution was added to 200 mL of reaction system in the form of supernatant. The initial amounts of the four enzymes added 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-valine (D,L-NVA), 300 mM ammonium formate, 0.2 mM NAD+, pH 8.0, temperature 40 ℃, and the reaction was carried out under oxygen-provided conditions for 6 h. Samples were taken at 1-h intervals to detect the substrate and product.

[0115] The catalytic system for D,L-ABA substrates differs from that for D,L-valine (D,L-NVA) substrates in that D,L-ABA is used instead of D,L-NVA; otherwise, they are identical.

[0116] Intermediate product HPLC detection conditions: Column type: 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.

[0117] Substrate and final product were determined by pre-column derivatization in liquid chromatography: Derivatization reagent preparation: Weigh 0.03 g of phthalaldehyde and 0.1 g of N-acetyl-L-cysteine, add 400 μL of anhydrous ethanol and 4 mL of borate buffer, sonicate until completely dissolved, and use immediately. Derivatization reaction and determination: Add 100 μL of derivatization reagent to 100 μL of sample, mix well, and incubate at 25℃ for 5 min. Chromatographic conditions: Column type: Pntulips® QS-C18, 5 μm, 4.6 mm × 250 mm; Detection wavelength: 334 nm; Column temperature: 30℃; Injection volume: 20 μL; Flow rate: 1 mL / min; Mobile phase: 50 mM sodium acetate aqueous solution: methanol = 50:50.

[0118] Figure 9 The graph shows the changes in the concentrations of substrates, intermediates, and final products over time during the four-enzyme cascade preparation of L-n-valine and L-2-aminobutyric acid. The substrates are D,L-n-valine (D-NVA) and D,L-2-aminobutyric acid (D-ABA); the intermediates are 2-pentanone and 2-butanone; and the final products are L-n-valine (L-NVA) and L-2-aminobutyric acid (L-ABA).

[0119] from Figure 9 As can be seen from the HPLC results, the concentrations of the final products L-valine and L-2-aminobutyric acid can reach 199 mM, and the ee value reaches 99%.

[0120] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

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

1.

2. The mutant as described in claim 1, characterized in that, The mutant is a substitution of V53F and Y242V.

3. An isolated polynucleotide, characterized in that, Encode the mutant as described in claim 1 or 2.

4. A nucleic acid construct, characterized in that, It contains the polynucleotide as described in claim 3.

5. A host cell, characterized in that, It includes the nucleic acid construct as described in claim 4 or the genome in which the polynucleotide as described in claim 3 is integrated.

6. A composition, characterized in that, It includes the mutant as described in claim 1 or 2.

7. The composition according to claim 6, characterized in that, It also contains catalase, glutamate dehydrogenase, and formate dehydrogenase.

8. The use of the mutant of claim 1, the polynucleotide of claim 3, the nucleic acid construct of claim 4, the host cell of claim 5, or the composition of claim 6 in the preparation of 2-pentanone, 2-butanone, L-valine, or L-2-aminobutyric acid.

9. A method for preparing 2-pentanone acid or 2-butanone acid, characterized in that, The substrate is catalyzed using the mutant as described in claim 1, the polynucleotide as described in claim 3, the nucleic acid construct as described in claim 4, the host cell as described in claim 5, or the composition as described in claim 6, wherein the substrate is selected from D,L-valine or D,L-2-aminobutyric acid.

10. A method for preparing L-valine or L-2-aminobutyric acid, characterized in that, The substrate is catalyzed using the mutant as described in claim 1, the polynucleotide as described in claim 3, the nucleic acid construct as described in claim 4, the host cell as described in claim 5, or the composition as described in claim 6, wherein the substrate is selected from D,L-valine or D,L-2-aminobutyric acid.

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