D-amino acid oxidase mutants and their use in the preparation of l-glufosinate

Abstract

The present application relates to a kind of D-amino acid oxidase mutant with significantly improved catalytic performance, coding gene, vector containing coding gene, genetically engineered bacteria, and the mutant is applied in the microbial catalytic preparation of L-glufosinate-ammonium.The D-amino acid oxidase mutant is obtained by single-point mutation or multiple-point combined mutation of the amino acid shown in SEQ ID NO.1.The beneficial effects of the present application mainly include: the present application provides D-amino acid oxidase mutant with improved enzyme activity and enzyme thermal stability, which can be used for microbial catalytic preparation of L-glufosinate-ammonium, conducive to industrial production, and has good application prospect.

Description

(I) Technical Field

[0001] This invention relates to a D-amino acid oxidase mutant (DAAO), its encoding gene, a vector containing the encoding gene, genetically engineered bacteria, and their application in the microbial catalytic preparation of L-glufosinate. (II) Background Technology

[0002] Phosphinothricin (PPT), chemically known as 2-amino-4-[hydroxy(methyl)phosphono]butyric acid, is the world's second most widely used herbicide for genetically modified crops. It has excellent herbicidal performance and relatively few phytotoxic side effects, and has huge market potential in the future.

[0003] PPT has two optical isomers: L-PPT and D-PPT; however, only the L-type possesses herbicidal activity. Commercially available PPT is generally a racemic mixture. If PPT products could be used in the pure L-configuration optical isomer, the amount of PPT used could be significantly reduced, which is of great importance for improving atom economy, reducing usage costs, and alleviating environmental pressure.

[0004] Currently, the most widely used method for removing D-PPT is to use D and L-PPT as raw materials, catalyzing D-PPT with DAAO to obtain the L-PPT precursor 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO), which is then catalyzed by amino acid dehydrogenases or transaminases to obtain L-PPT. Due to the high selectivity of DAAO, this method can not only effectively remove D-PPT, but its key intermediate PPO can also be further converted into L-PPT, effectively improving atom utilization. Therefore, screening for high-yield PPO mutants is particularly important. (III) Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies, such as low enzyme activity and poor enzyme thermal stability of DAAO, and to provide a D-AAO mutant with significantly improved catalytic performance, the encoding gene, a vector containing the encoding gene, genetically engineered bacteria, and its application in the microbial catalytic preparation of L-PPT.

[0006] The technical solution adopted in this invention is:

[0007] A DAAO mutant is obtained by single-point or multi-point combined mutation of amino acid positions 54, 58, 213, 239, 73, 77, 79, 147, 185, 43, 45, 206, 207, 215, 122, 132, 195, and 234 of the amino acid sequence shown in SEQ ID NO.1.

[0008] The amino acid sequence of D-AAO, containing the sequence shown in SEQ ID NO.1, may be referred to in this application as the wild-type enzyme of DAAO. The nucleotide sequence of the wild-type enzyme may be the nucleotide sequence shown in SEQ ID NO.8. SEQ ID NO.1 is the amino acid sequence of DAAO derived from Rhodotorula taiwanensis. SEQ ID NO.8 is the nucleotide sequence of DAAO derived from Rhodotorula taiwanensis.

[0009] Preferably, the mutation is one of the following: (1) the 54th amino acid residue N is mutated to V, D or T, the 58th amino acid residue F is mutated to H, R, K or Q, and the 213th amino acid residue M is mutated to S, N or R; (2) the 54th amino acid residue N is mutated to V, D or T, the 58th amino acid residue F is mutated to H, R, K or Q, the 213th amino acid residue M is mutated to S, N or R, and the 239th amino acid residue L is mutated to E, D or G. Or Q; (3) The 54th amino acid residue N is mutated to V, D or T, the 58th amino acid residue F is mutated to H, R, K or Q, the 213th amino acid residue M is mutated to S, N or R, the 239th amino acid residue L is mutated to E, D, G or Q, the 73rd amino acid residue A is mutated to L, the 77th amino acid residue Q is mutated to W, the 79th amino acid residue V is mutated to M, the 147th amino acid residue Q is mutated to M, and the 185th amino acid residue S mutates to M; (5) N mutates to V, D or T at position 54, F mutates to H, R, K or Q at position 58, M mutates to S, N or R at position 213, L mutates to E, D, G or Q at position 239, A mutates to S at position 43, T mutates to M at position 45, S mutates to A at position 206, D mutates to P at position 207, and M mutates to M at position 215. (6) Amino acid residue S is mutated to F; amino acid residue N at position 54 is mutated to V, D or T; amino acid residue F at position 58 is mutated to H, R, K or Q; amino acid residue M at position 213 is mutated to S, N or R; amino acid residue L at position 239 is mutated to E, D, G or Q; amino acid residue E at position 122 is mutated to P; amino acid residue Y at position 132 is mutated to F; amino acid residue E at position 195 is mutated to Y; amino acid residue C at position 234 is mutated to L.

[0010] More preferably, the amino acid sequence of the mutant is shown in one of SEQ ID NO.2 to 7.

[0011] SEQ ID NO.2 is the amino acid sequence of DAAO mutant I (M213S / F58H / N54V).

[0012] SEQ ID NO.3 is the amino acid sequence of DAAO mutant II (M213S / F58H / N54V / L239G).

[0013] SEQ ID NO.4 is DAAO mutant III (M213S / F58H / N54V / L239G)

[0014] The amino acid sequence is / A73L / Q77W / V79M / Q147M / S185M.

[0015] SEQ ID NO.5 is DAAO mutant IV (M213S / F58H / N54V / L239G)

[0016] The amino acid sequence is ( / A43S / T45M / S206A / D207P / S215F).

[0017] SEQ ID NO.6 is the DAAO mutant V (M213S / F58H / N54V / L239G).

[0018] The amino acid sequence of / E122P / Y132F / E195Y / C234L).

[0019] SEQ ID NO.7 is the DAAO mutant VI (M213S / F58H / N54V / L239G).

[0020] The amino acid sequence is / A73L / Q77W / V79M / Q147M / S185M / A43S / T45M / S206A / D207P / S215FE122P / Y132F / E195Y / C234L.

[0021] The present invention also relates to the gene encoding the DAAO mutant and the recombinant vector.

[0022] The recombinant expression vector is constructed by linking the nucleic acid encoding the DAAO mutant gene of the present invention to various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, such as commercially available plasmids, granules, bacteriophages, or viral vectors, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the DAAO. The preferred vector is a plasmid, more preferably plasmid pET28a.

[0023] The present invention also relates to genetically engineered bacteria containing a gene encoding the DAAO mutant described above.

[0024] Recombinant expression transformants can be prepared by transforming a pre-constructed recombinant expression vector into host cells. The host cells can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate spontaneously and effectively express the target protein after induction with an inducer. This invention preferentially uses *Escherichia coli* as the host cell, and more preferably *E. coli* BL21(DE3) for efficient expression of the DAAO mutant described in this invention.

[0025] This invention also relates to the application of the DAAO mutant in the microbial catalytic preparation of L-PPT.

[0026] The reaction formulas involved are as follows:

[0027]

[0028] The beneficial effects of this invention are mainly reflected in the following aspects: This invention provides a DAAO mutant with improved enzyme activity and enzyme thermostability, which can be used for microbial catalysis to prepare L-PPT, which is conducive to industrial production and has good application prospects. (IV) Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments:

[0030] The plasmid extraction kit and DNA purification and recovery kit used in the examples were purchased from Hangzhou Qingke Zixi Biotechnology Co., Ltd.; the one-step cloning kit was purchased from Novizan Biotechnology Co., Ltd.; E. coli BL21(DE3), plasmid pET-24a(+), and whole gene synthesis were performed by Sangon Biotech (Shanghai) Co., Ltd.; DNA labeling, low molecular weight standard protein, and protein precast gel were purchased from Beijing GenStar Co., Ltd.; the ClonExpress II OneStep Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; pfu DNA polymerase and DpnI endonuclease were purchased from Thermo Fisher Scientific (China) Co., Ltd.; primer synthesis and sequence sequencing were performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0031] The reagents D and L-PPT used in the downstream catalytic process were purchased from Sigma-Aldrich; 2,4-dinitrophenylhydrazine (DNPH) was purchased from Aladdin Reagents (Shanghai, China); and commercially available micrococcal catalase was purchased from Sigma-Aldrich (Shanghai, China). Other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0032] Other experimental methods in the examples that do not specify specific conditions were performed in accordance with conventional methods and conditions, or as selected according to the product instructions.

[0033] The following examples demonstrate the detection and analysis of products using high-performance liquid chromatography (HPLC).

[0034] The HPLC analytical method is as follows: chromatographic column / C18; Column temperature / 30℃; Flow rate / 1mL / min; Detection wavelength / 232nm; Mobile phase: 50mM(NH2)HPO4, with 1% of 10% tetrabutylammonium bromide aqueous solution added, pH adjusted to 3.8 with phosphoric acid, and 12% acetonitrile added.

[0035] The content of two configurations of PPT was examined using a chiral HPLC method. Specifically, the chiral HPLC method was as follows: column: Pntulips QS-C18; mobile phase: 50 mM ammonium acetate solution: methanol = 9:1; detection wavelength: 338 nm; flow rate: 1 mL / min; column temperature: 30 °C.

[0036] Derivatization reagent: Weigh 0.1 g of o-phthalaldehyde and 0.12 g of N-acetyl-L-cysteine, respectively, and dissolve them in 10 ml of ethanol. Add 40 ml of 0.1 M borate buffer (pH 9.8). Shake to dissolve completely and store at 4°C for later use (not exceeding 3 days). Derivatization reaction and determination: Take 200 μL of sample and add 400 μL of derivatization reagent. Mix well and incubate at 30°C for 5 min. Add 400 μL of ultrapure water and mix. Inject 10 μL for analysis.

[0037] Example 1: Preparation of genetically engineered bacteria

[0038] The wild-type DAAO (wtDAAO) gene sequence from *Rhodotorula taiwanensis* (GenBank ID: POY70719.1), with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.8, was synthesized and inserted into the expression plasmid pET-24a(+), yielding pET-24a(+)-DAAO. After sequencing verification, pET-24a(+)DAAO was transformed into the expression host *Escherichia coli* BL21(DE3) for subsequent recombinase expression.

[0039] LB liquid culture medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in water and brought to a final volume, sterilized at 121℃ for 20 min, ready for use.

[0040] After activation by streak plating, the engineered bacteria with confirmed sequencing results were inoculated into 10 ml of LB broth containing 50 μg / ml kanamycin. The culture was incubated at 37°C with shaking for 10–12 h. Then, a 2% inoculum was transferred to 100 ml of fresh LB broth containing 50 μg / ml kanamycin. The culture was incubated at 37°C with shaking until the OD600 reached approximately 0.8. The temperature was then lowered to 30°C, and IPTG was added to a final concentration of 0.5 mM. Induction culture was continued for 16 h. After incubation, the culture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored at -20°C for later use. The collected bacterial cells were washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL of pH 8.0 phosphate buffer, homogenized, and the lysate was centrifuged to remove the precipitate, yielding a crude enzyme solution containing recombinant wtDAAO enzyme.

[0041] Example 2: Construction of DAAO mutant I (positions 54, 58, and 213)

[0042] Based on the wild-type DAAO sequence described in Example 1, positions 213, 58, and 54 were mutated. Primer sequences for PCR were designed for the mutant DAAO sequence, targeting positions 213, 58, and 54, and are shown in Tables 1, 2, and 3 according to the mutation sequence of the mutation sites.

[0043] Table 1

[0044] Serial number Primer name Primer sequence 1 213A F TGCAAACGTTGCACCGCTGACAGCAGCGAT 2 213R F TGCAAACGTTGCACCCGTGACAGCAGCGAT 3 213N F TGCAAACGTTGCACCAACGACAGCAGCGAT 4 213D F TGCAAACGTTGCACCGATGACAGCAGCGAT 5 213C F TGCAAACGTTGCACCTGTGACAGCAGCGAT 6 213Q F TGCAAACGTTGCACCCAAGACAGCAGCGAT 7 213E F TGCAAACGTTGCACCGAAGACAGCAGCGAT 8 213G F TGCAAACGTTGCACCGGTGACAGCAGCGAT 9 213H F TGCAAACGTTGCACCCATGACAGCAGCGAT 10 213I F TGCAAACGTTGCACCATCGACAGCAGCGAT 11 213L F TGCAAACGTTGCACCTTGGACAGCAGCGAT 12 213K F TGCAAACGTTGCACCAAGGACAGCAGCGAT 13 213F F TGCAAACGTTGCACCTTCGACAGCAGCGAT 14 213P F TGCAAACGTTGCACCCCTGACAGCAGCGAT 15 213S F TGCAAACGTTGCACCTCTGACAGCAGCGAT 16 213T F TGCAAACGTTGCACCACTGACAGCAGCGAT 17 213W F TGCAAACGTTGCACCTGGGACAGCAGCGAT 18 213Y F TGCAAACGTTGCACCTACGACAGCAGCGAT 19 213V F TGCAAACGTTGCACCGTTGACAGCAGCGAT 20 213R GGTGCAACGTTTGCAATCGCTCTTCACCAG

[0045] Table 2

[0046]

[0047]

[0048] Table 3

[0049] Serial number Primer name Primer sequence 1 54A F TGGGCGGGTGCGGCTTGGACCCCGCAAAT 2 54R F TGGGCGGGTGCGCGTTGGACCCCGCAAAT 3 54D F TGGGCGGGTGCGGATTGGACCCCGCAAAT 4 54C F TGGGCGGGTGCGTGTTGGACCCCGCAAAT 5 54Q F TGGGCGGGTGCGCAATGGACCCCGCAAAT 6 54E F TGGGCGGGTGCGGAATGGACCCCGCAAAT 7 54G F TGGGCGGGTGCGGGTTGGACCCCGCAAAT 8 54H F TGGGCGGGTGCGCATTGGACCCCGCAAAT 9 54I F TGGGCGGGTGCGATTTGGACCCCGCAAAT 10 54L F TGGGCGGGTGCGTTATGGACCCCGCAAAT 11 54K F TGGGCGGGTGCGAAATGGACCCCGCAAAT 12 54M F TGGGCGGGTGCGATGTGGACCCCGCAAAT 13 54F F TGGGCGGGTGCGTTTTGGACCCCGCAAAT 14 54P F TGGGCGGGTGCGCCTTGGACCCCGCAAAT 15 54S F TGGGCGGGTGCGTCTTGGACCCCGCAAAT 16 54T F TGGGCGGGTGCGACTTGGACCCCGCAAAT 17 54W F TGGGCGGGTGCGTGGTGGACCCCGCAAAT 18 54Y F TGGGCGGGTGCGTATTGGACCCCGCAAAT 19 54V F TGGGCGGGTGCGGTTTGGACCCCGCAAAT 20 54R CGCACCCGCCCACGGGCTCGCAAAGGTTT

[0050] The PCR (25 μL) amplification system is as follows:

[0051] 12.5 μL of 2×PCR buffer, 0.5 μL each of forward and reverse primers, 0.5 μL of template plasmid, 0.5 μL of dNTPs, 0.5 μL of high-fidelity enzyme, and ddH2O to bring the total to 25 μL.

[0052] The PCR amplification procedure is as follows:

[0053] (1) Pre-denaturation at 95℃ for 5 min, (2) Denaturation at 95℃ for 30 seconds, (3) Annealing at 60℃ for 30 seconds, (4) Extension at 72℃ for 5 min, 30 cycles, (5) Extension at 72℃ for 10 min, (6) Storage at 4℃.

[0054] After PCR, 5 μL of the amplified product was analyzed by nucleic acid gel electrophoresis. 0.5 μL of Dpn I restriction enzyme was added to the PCR product with a clear target band, and the sample was digested at 37°C for 1 hour. After the reaction, the product was transformed into BL21 competent cells, plated on LB medium containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Transformants containing the mutant were harvested. Bacterial cells were obtained according to Example 1.

[0055] Example 3: High-throughput screening of mutant libraries

[0056] The screening was conducted following these experimental steps:

[0057] The transformants obtained in Example 2 were inoculated into 96-well plates and cultured in a constant temperature shaker at 37°C for 12-16 hours at a shaker speed of 200 rpm. The seed culture from the 96-well plates was then transferred to 96-well fermentation medium, and cultured at OD... 600 Add IPTG inducer when the pH is 0.4–0.7, and incubate in a constant temperature shaker at 28°C for 12–16 hours at a shaking speed of 200 rpm. Centrifuge the cultured 96-well fermentation broth at 4000 rpm for 10 minutes, discard the supernatant, and collect the bacterial cells. React the collected bacterial cells with a certain concentration of D,L-PPT in a 96-well plate for 1 hour, then centrifuge at 4000 rpm for 10 minutes.

[0058] Colorimetric reaction: The supernatant of the centrifuged reaction solution was pipetted into a 96-well clear plate, and 2 mM 2,4-dinitrophenylhydrazine reagent was added. The mixture was then thoroughly mixed using a pipette and incubated at 37°C for 20 minutes. After the reaction, 100 μL of 1 M NaOH was added, and the mixture was stirred for 30 seconds, resulting in a reddish-brown compound. The absorbance at 380 nm was detected using a high-throughput microplate reader. Positive clones (M213N, M213R, M213S), (M213S / F58H, M213S / F58R, M213S / F58K, M213S / F58Q) were obtained by screening with wtDAAO as a control.

[0059] (M213S / F58H / N54V, M213S / F58H / N54D, M213S / F58H / N54T).

[0060] Example 4: Comparison of enzyme activity of DAAO mutant I

[0061] The positive clones (M213N, M213R, M213S), (M213S / F58H, M213S / F58R, M213S / F58K, M213S / F58Q), and (M213S / F58H / N54V, M213S / F58H / N54D, M213S / F58H / N54T) obtained in Example 3 were re-screened. The re-screening reaction was carried out by detecting the catalytic efficiency of the mutants. Specifically, the catalytic efficiency of DAAO and its mutants was compared by measuring the amount of PPO generated using HPLC. The 1 ml reaction system included: 50 mM racemic PPT ammonium salt, 50 mM pH 8.0 phosphate buffer, 8000 U / L catalase, and 50 g / L DAAO or its mutant frozen stem cells. After 2 hours of reaction, a sample of the reaction solution was taken for processing, the concentration of PPO was measured, and the conversion rate (product PPO concentration / initial substrate D,L-PPT concentration × 100%) was calculated, as shown in Table 4.

[0062] Table 4

[0063] Enzyme number Wild type / mutant Product PPO / mM Conversion rate (%) 1 wtDAAO 0 0.12 2 M213S 0.287±0.05 1.15 3 M213R 0.126±0.02 0.5 4 M213N 0.189±0.03 0.75 5 M213S / F58H 3.58±0.05 14.38 6 M213S / F58R 2.74±0.03 10.96 7 M213S / F58K 2.69±0.02 10.76 8 M213S / F58Q 1.62±0.03 6.32 9 M213S / F58H / N54V 8.35±0.04 33.4 10 M213S / F58H / N54D 7.68±0.05 30.72 11 M213S / F58H / N54T 8.13±0.02 32.52

[0064] Example 5: Construction of the DAAO mutant (position 239)

[0065] Based on the mutant sequence (N54V / F58Q / M213S) described in Example 4, error-prone PCR (epPCR) was performed. Primer sequences designed for error-prone PCR are shown in Table 5.

[0066] Table 5

[0067] Serial Number Primer name Primer sequence 1 epPCR-F ATGGCGCCGAGCAAGCGTGTGGTTGTG 2 epPCR-N TCAGTGGTGGTGGTGGTGGTGCTCGAGT

[0068] The epPCR (30 μL) amplification system was as follows: Using a transient error-prone PCR kit, under the conditions of 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2, and 0.25 mM MnCl2, 1 μL of template plasmid and 0.5 μL each of forward and reverse primers were added to a 30 μL reaction mixture. The PCR amplification conditions were: 95℃ for 5 min, (90℃ for 30 s, 55℃-65℃ for 30 s, 72℃ for 5 min) × 30 cycles, followed by 72℃ for 10 min.

[0069] After PCR, gel electrophoresis analysis was performed as described in Example 2, the template was digested, and transformation was performed. Then, bacterial cells were obtained as described in Example 1. Finally, positive clones were screened as described in Example 3. A mutant at position 239 (specifically L239G) was obtained.

[0070] Example 6: Comparison of enzyme activities of DAAO mutant II

[0071] As described in Example 4, the positive clones obtained from Example 5 were subjected to combined rescreening, and the catalytic efficiencies among the mutants were compared, as detailed in Table 6.

[0072] Table 6

[0073] Enzyme number mutant Product PPO / mM Conversion rate (%) 9 I(M213S / F58H / N54V) 8.39±0.04 33.4 12 II(M213S / F58H / N54V / L239G) 12.56±0.04 50.24

[0074] As can be seen from the table above, the activity of mutant II (M213S / F58H / N54V / L239G) is significantly improved. Next, further mutations to improve thermal stability will be carried out based on mutant II.

[0075] Example 7: Construction of DAAO mutant III and comparison of enzyme activity and thermostability

[0076] Based on the mutant II sequence described in Example 6, positions 73, 77, 79, 147, and 185 were mutated, specifically resulting in position III (M213S / F58H / N54V / L239G / A73L / Q77W / V79M / Q147M / S185M). Primer sequences for mutant PCR were designed targeting these sites in mutant III, as shown in Table 7.

[0077] Table 7

[0078] Serial Number Primer name Primer sequence 1 A73L F TGGGAGACCTTAACCTTTAACCAGTGGGTTG 2 A73L R GGTTAAAGGTTAAGGTCTCCCATTTCGCCTG 3 77W / 79M F GACCTTTAACTGGTGGATGGATCTGGTGCCGCAAG 4 77W / 79M R GCACCAGATCCATCCACCAGTTAAAGGTCGCGGTCTC 5 Q147M F GTTCTGCCAGTATCTGATGCGTGAAGGCCAG 6 Q147M R GCGCTTCACGATGCAGATACTGGCAGAACT 7 S185M F GGGTGCGAAGATGATTGCGGGTGTTGAAGA 8 S185M R ACACCCGCAATCATCTTCGCACCCAGACCGGT

[0079] The PCR amplification system and conditions were the same as described in Example 2, and bacterial cells were obtained as described in Example 1. The catalytic efficiency of the mutants was detected as described in Example 4. The thermostability of the mutants was tested by measuring residual enzyme activity after incubation at a certain temperature for a certain time. Specifically, the residual enzyme activity was measured after incubation at 45°C for 30 minutes and then reacted at 30°C for 1 hour. The catalytic efficiency and thermostability effects between the single-point mutations and combined mutants are shown in Table 8.

[0080] Table 8

[0081]

[0082] Example 8: Construction of DAAO mutant IV and comparison of enzyme activity and thermostability

[0083] Based on the mutant II sequence described in Example 6, positions 43, 45, 112, 206, 207, and 215 were mutated, specifically (M213S / F58H / N54V / L239G / A43S / T45M / S206A / D207P / S215F). Mutant PCR primer sequences were designed targeting these sites in mutant IV, as shown in Table 9.

[0084] Table 9

[0085] Serial number Primer name Primer sequence 1 43 / 45F GATACCGTTTCTCAAATGTTTGCGAGCCCGTGG 2 43 / 45R GCTCGCAAACATTTGAGAAACGGTATCTTCCGGC 3 206 / 207F TCTGGTGAAGGCTCCTTGCAAACGTTGCACCTCT 4 206 / 207R ACGTTTGCAAGGAGCCTTCACCAGAACGGTTTG 5 215F CACCTCTGACTTTAGCGATCCGAACAGCC 6 215R CGGATCGCTAAAGTCAGAGGTGCAACGT

[0086] The PCR amplification system and conditions were the same as those described in Example 2, and bacterial cells were obtained as described in Example 1. The catalytic efficiency of the mutants was detected as described in Example 4. The thermostability of the mutants was tested by measuring residual enzyme activity after incubation at a certain temperature for a certain time. Specifically, the residual enzyme activity was measured after incubation at 45°C for 30 minutes and then reacted at 30°C for 1 hour. The catalytic efficiency and thermostability effects between the single-point mutations and combined mutants are shown in Table 10.

[0087] Table 10

[0088]

[0089] Example 9: Construction of DAAO mutant V and comparison of enzyme activity and thermostability

[0090] Based on the mutant II sequence described in Example 6, positions 122, 132, 195, and 234 were mutated, specifically (M213S / F58H / N54V / L239G / E122P / Y132F / E195Y / C234L). Primer sequences for mutant PCR targeting these sites in mutant V were designed, as shown in Table 11.

[0091] Table 11

[0092] Serial number Primer name Primer sequence 1 122F GAGAGCAGCCCTTGCCCGCCGGGTGCGATT 2 122R CCGGCGGGCAAGGGCTGCTCTCCAGTTTAC 3 132F GGTGTTACCTTTGATACCCTGAGCGTGAAC 4 132R CAGGGTATCAAAGGTAACACCAATCGCAC 5 195F CAGGAAGTGTATCCGATTCGTGGCCAAAC 6 195R CACGAATCGGATACACTTCCTGGTCTTCAAC 7 234F CGAGGTGATCTTAGGTGGCACCTACCTG 8 234R GGTGCCACCTAAGATCACCTCGCCACC

[0093] The PCR amplification system and conditions were the same as those described in Example 2, and bacterial cells were obtained as described in Example 1. The catalytic efficiency of the mutants was detected as described in Example 4. The thermostability of the mutants was tested by measuring residual enzyme activity after incubation at a certain temperature for a certain time. Specifically, the residual enzyme activity was measured after incubation at 45°C for 30 minutes and then reacted at 30°C for 1 hour. The catalytic efficiency and thermostability effects between the single-point mutations and combined mutants are shown in Table 12.

[0094] Table 12

[0095]

[0096] Example 10: Construction of DAAO mutant VI and comparison of enzyme activity and thermostability

[0097] The above-mentioned DAAO mutant IV was combined with mutations to obtain mutant VI.

[0098] (M213S / F58H / N54V / L239G / A73L / Q77W / V79M / Q147M / S185M / A43S / T45M / S206A / D207P / S215F / E122P / Y132F / E195Y / C234L). The PCR amplification system and conditions were the same as described in Example 2, and bacterial cells were obtained as described in Example 1. The catalytic efficiency of the combined mutants II, III, IV, V, and VI was tested as described in Example 4. The thermostability of the mutants was tested by measuring the residual enzyme activity after incubation at a certain temperature for a certain time. Specifically, the residual enzyme activity was measured after incubation at 50℃ for 15 minutes and 55℃ for 15 minutes, followed by reaction at 30℃ for 1 hour. The catalytic efficiency and thermostability of the combined mutants are shown in Table 13.

[0099] Table 13

[0100]

[0101]

[0102] As can be seen from the table above, mutant VI exhibits significantly improved thermostability without affecting enzyme activity. Clearly, the above examples are merely illustrative and not intended to limit the implementation. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. A D-amino acid oxidase mutant, characterized in that The mutant amino acid sequence is shown in one of SEQ ID NO. 4~7.

2. A gene encoding the D-amino acid oxidase mutant of claim 1.

3. A recombinant vector containing a gene encoding the D-amino acid oxidase mutant of claim 1.

4. A genetically engineered bacterium containing a gene encoding the D-amino acid oxidase mutant of claim 1.

5. Use of the D-amino acid oxidase mutant of claim 1 in the microbial catalytic preparation of L-glufosinate.

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