D-amino acid oxidase with improved activity and its use in the synthesis of spinosyn

Abstract

The application discloses a D-amino acid oxidase with improved enzyme activity and application thereof in synthesis of pure glufosinate-ammonium, and belongs to the technical field of microbial synthesis. The application provides a D-amino acid oxidase mutant A51V with significantly improved enzyme activity, wherein under the reaction condition of 30 DEG C and pH 8.0, the enzyme activity of the mutant can reach 1.29 times of that of a wild-type enzyme after 3 h of catalytic conversion, the efficiency of catalyzing D-glufosinate to generate PPO is significantly improved, the mutant is more suitable for the demand of large-scale industrial production of optically pure glufosinate-ammonium compared with the wild-type enzyme, the production cycle and production cost of resolving racemic D,L-glufosinate can be effectively reduced, waste emission in the production process is reduced, and the environmental burden is reduced, and the application has important application value for the biological synthesis industry of glufosinate-ammonium.

Description

Technical Field

[0001] This invention relates to a D-amino acid oxidase with enhanced enzyme activity and its application in the synthesis of chlorpyrifos, belonging to the field of microbial synthesis technology. Background Technology

[0002] Glufosinate (PPT) is a phosphorus-containing, non-protein-derived amino acid, a broad-spectrum, highly effective, and non-selective herbicide. Commercially available glufosinate products typically consist of a mixture of two enantiomers, with only the L-enantiomer exhibiting herbicidal activity. Currently, refined glufosinate is mainly synthesized asymmetrically using chiral substrates or via racemic reactions using racemic glufosinate as a substrate. The use of racemic glufosinate has led to high costs and severe environmental impact, thus the development of efficient methods for producing optically pure refined glufosinate has attracted widespread attention. It is derived from *Rhodotorula glutinis* (…). Rhodosporidium toruloides The D-amino acid oxidase (EC: 1.4.3.3) catalyzes the oxidation of D-glufosinate to 2-oxo-4-[(hydroxy)(-methyl)phosphono]butyric acid (PPO), exhibiting the strongest relative enzyme activity at 30℃ and pH 8.0, reaching a relative enzyme activity of [value missing]. Furthermore, the increasing demand for glufosinate-ammonia oxidase places higher demands on the bioracemicization reaction of glufosinate-ammonia oxidase; therefore, screening for D-amino acid oxidases with higher enzyme activity has significant application value for glufosinate-ammonia oxidase production. Summary of the Invention

[0003] This invention provides a D-amino acid oxidase mutant derived from Rhodotorula buergerianum, wherein the D-amino acid oxidase mutant is based on the D-amino acid oxidase shown in SEQ ID NO.1, with alanine at position 51 mutated to valine.

[0004] In one embodiment, the D-amino acid oxidase mutant is obtained by mutating glutamine at position 335 to glycine based on the D-amino acid oxidase shown in SEQ ID NO.1, to obtain the D-amino acid oxidase mutant Q335G having the amino acid sequence shown in SEQ ID NO.2.

[0005] The present invention also provides a gene encoding the D-amino acid oxidase mutant.

[0006] In one embodiment, the gene encoding the D-amino acid oxidase mutant has a nucleotide sequence as shown in SEQ ID NO.3.

[0007] The present invention also provides a recombinant plasmid carrying the said gene.

[0008] In one embodiment, the recombinant plasmid uses the pET series as a vector.

[0009] In one embodiment, the pET series includes pET-28a.

[0010] The present invention also provides a D-amino acid oxidase mutant expressing the D-amino acid oxidase, or a recombinant cell carrying the gene or containing the recombinant plasmid.

[0011] In one embodiment, the recombinant cells are bacteria or fungi as host cells; In one embodiment, the bacteria are selected from Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum, and the fungus is yeast.

[0012] Preferably, Escherichia coli is used as the host cell.

[0013] In one embodiment, the Escherichia coli includes, but is not limited to, Escherichia coli BL21 (DE3).

[0014] The present invention also provides a recombinant enzyme catalyst containing the D-amino acid oxidase mutant, which is any one of the following forms: (1) Culture the recombinant cells and isolate cells containing the mutant; (2) Culture the recombinant cells, separate and break the cells to obtain a cell lysate containing the mutant; (3) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate.

[0015] The present invention also provides a method for improving the activity of D-amino acid oxidase, wherein the method is based on the D-amino acid oxidase shown in SEQ ID NO.1, by mutating alanine at position 51 to valine.

[0016] The present invention also provides a genetically engineered bacterium that expresses the D-amino acid oxidase mutant.

[0017] In one embodiment, the genetically engineered bacteria also express at least one coenzyme associated with the generation of PPO.

[0018] In one embodiment, the coenzyme includes catalase.

[0019] The present invention also provides a method for resolving racemic D,L-glufosinate, wherein the method uses D,L-glufosinate as a substrate and the recombinant cells, the recombinase catalyst, or the genetically engineered bacteria as a catalyst to resolve racemic D,L-glufosinate and generate intermediate PPO.

[0020] In one embodiment, the catalytic system further includes catalase; the catalase can be any enzyme known in the art that has catalase activity.

[0021] In one embodiment, the catalytic temperature is 25-40°C and the pH is 7-8.

[0022] The present invention also provides the application of the D-amino acid oxidase mutant, or the gene or recombinant vector, or the recombinant cell, or the recombinase catalyst, or the genetically engineered bacteria, or the method in any of the following: (1) Preparation of L-glufosinate; (2) Preparation of intermediate PPO.

[0023] [Beneficial Effects] This invention provides a D-amino acid oxidase mutant with enhanced enzyme activity, which can increase the enzyme activity of mutant A51V to 1.29 times that of wild-type enzyme after transformation at 30°C, pH 8.0 and 3 h, thus helping to broaden its application scope in the pharmaceutical field. Attached Figure Description

[0024] Figure 1 D-amino acid oxidase SDS-PAGE verification image. Lane 1 is the intracellular supernatant of BL21(DE3) / pET-28a-Daao; Figure 2 Relative enzyme activity of D-amino acid oxidase at different pH and temperature; Figure 3 Relative enzymatic activities of different D-amino acid oxidase mutants; Figure 4 The conversion results in a 5 L bioreactor. Detailed Implementation

[0025] The following embodiments involve Escherichia coli JM109 and BL21(DE3) were both purchased from Sangon Biotech (Shanghai) Co., Ltd.; the pET-28a plasmid involved in the following examples was purchased from BioVector China Plasmid Vector Strains Cell Gene Depository Center.

[0026] The materials involved in the following embodiments are as follows: 1. Culture medium and buffer solution (1) LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0027] (2) LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L.

[0028] (3) TY fermentation medium: yeast extract 8 g / L, peptone 12 g / L, K3PO4 4.02 g / L, NaCl 3 g / L, citric acid monohydrate 2.1 g / L, ferric ammonium citrate 0.3 g / L, (NH4)2SO4 2.5 g / L, MgSO4•7H2O 0.5 g / L, pH 7.2.

[0029] (4) Feeding medium: 400 g / L glycerol, 50 g / L yeast extract, 25 g / L peptone.

[0030] The detection methods involved in the following embodiments are as follows: (1) Enzyme activity of D-amino acid oxidase: The concentration of the product was calculated using the addition reaction of 2,4-dinitrophenylhydrazine with aldehydes and ketones.

[0031] The D-amino acid oxidase activity assay system consisted of 3.6 mL of solution. 1.2 mL of PBS buffer (0.05 mM, pH 7.5) was added to the test tube, followed by 100 μL of 0.2 MD,L-glufosinate, then 100 μL of 400 U / mL commercial catalase. Finally, 20 μL of the appropriately diluted sample was added, mixed thoroughly, and the dilution factor was recorded. The reaction was initiated by placing the test tube in a 30°C constant-temperature shaking water bath. After 15 min of reaction time, 480 μL of 2 mg / mL 2,4-dinitrophenylhydrazine (prepared with 2 M hydrochloric acid) was immediately added and mixed to stop the D-amino acid oxidase reaction. The tube was then placed in a 37°C constant-temperature water bath for 10 min. 1.7 mL of 3 M NaOH was added for color development. The concentration of the product was detected by the addition reaction of 2,4-dinitrophenylhydrazine with aldehydes and ketones. The absorbance of the sample after the reaction was measured at 550 nm and recorded. Enzyme activity calculation is performed by using a standard curve plotted from the absorbance values ​​of different concentrations of PPO at 550 nm to calculate the product concentration in the sample after the oxidation reaction of D-amino acid oxidase.

[0032] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of 1 µmol of PPT to 1 µmol of PPO per minute under certain temperature and pH conditions. One enzyme activity unit is 1 U.

[0033] (2) HPLC method for detecting the enantiomers of D-PPT and L-PPT: The detector used was a fluorescence detector (FLD). After boiling 1 mL of the conversion sample for 5 min, the supernatant was collected by centrifugation at 12,000 rpm for 5 min. The sample was mixed with derivatization reagent (phthalaldehyde: N-acetylcysteine ​​= 10 mg: 12 mg, first dissolved in 1 mL of anhydrous ethanol, then diluted to 5 mL with 0.1 M sodium borate buffer at pH 9.8), reacted at 30 °C for 5 min, and then filtered through a 0.22 μm filter for HPLC detection.

[0034] HPLC method: The chromatographic column was a Dima Diamonsil C18(2), 4.6 mm × 250 mm, 5 μm. The mobile phase was 0.05 M ammonium acetate (pH=5.7): methanol = 9:1, the flow rate was 1 mL / min, the column temperature was 35℃, the emission wavelength was 350 nm, and the absorption wavelength was 450 nm.

[0035] (3) HPLC method for the detection of PPO: The detector used was a UV detector (VWD). 1 mL of the conversion sample was mixed with 10 μL of 6 M HCl, centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for HPLC detection.

[0036] HPLC method: The chromatographic column was a Dima Diamonsil C18(2), 4.6 mm × 250 mm, 5 μm. The mobile phase was 0.05 mM ammonium dihydrogen phosphate (pH=3.8):acetonitrile = 88:12, with 0.1% w / v tetrabutylammonium bromide added. The flow rate was 1 mL / min, the column temperature was 40℃, and the detection wavelength was 232 nm.

[0037] Example 1: Preparation of D-amino acid oxidase mutant plasmid (1) Construction of wild-type plasmid and strain of D-amino acid oxidase Synthesized from Rhodotorula buergerianum ( Rhodosporidium toruloides The D-amino acid oxidase gene Daao (nucleotide sequence shown in SEQ ID NO.4) was constructed into the pET-28a vector (between EcoRI and HandIII restriction sites), amplified using E. coli JM109, and screened by colony PCR and Sanger sequencing to obtain the recombinant plasmid pET-28a-Daao.

[0038] (2) Construction of D-amino acid oxidase mutant plasmid and strain Using the recombinant plasmid pET-28a-Daao prepared in step (1) as a template, primers containing the mutation site were designed, and whole-plasmid PCR was performed on the pET-28a-Daao plasmid using primers A51V-F / A51V-R to amplify the recombinant plasmid pET-28a-Daao containing the A51V mutation. A51V : pET-28a-Daao was subjected to full-plasmid PCR using primers A51V-F and A51V-R. The PCR product was purified and then used. Dpn The bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain Daao... A51V Mutant recombinant plasmid pET-28a-Daao A51V .

[0039] A51V-F:ttcgcttcaccatgggTCggcgcgACCtggacgcctttc; A51V-R:ccGAcccatggtgaagcgaaagtctggctcgagacgtcctc.

[0040] Example 2: Expression and purification of D-amino acid oxidase The recombinant plasmid pET-28a-Daao constructed in Example 1 and the mutant recombinant plasmid pET-28a-Daao were used. A51V Recombinant strains were obtained by transforming the bacteria into Escherichia coli BL21(DE3). E.Coli BL21 / pETDuet-Daao and E.Coli BL21 / pET-28a-Daao A51V .

[0041] The recombinant strain was inoculated into 10 mL of LB liquid medium and cultured at 37°C and 200 rpm for 12 h. Then, it was transferred to 100 mL of TY fermentation medium at a 1% (v / v) inoculation rate and cultured at 37°C for 2 h. Finally, IPTG was added to a final concentration of 0.8 mM and the culture was continued at 25°C and 200 rpm for 20 h to induce D-amino acid oxidase expression in the medium to OD. 600 It is 12.

[0042] After the culture was completed, the bacterial culture was collected, the cells were washed with phosphate buffer, centrifuged and the cells were collected, and the cells were sonicated: 400 w for 1 second, pause for 3 seconds, for 15 min. After sonication, the cells were centrifuged at 4℃ and 8,000 rpm for 20 min. The cell wall supernatant and cell wall precipitate were separated to obtain crude enzyme solutions of wild-type and mutant enzymes.

[0043] The crude wild-type enzyme was analyzed using SDS-PAGE, and the results are as follows: Figure 1 The molecular weight of the enzyme protein is approximately 40.1 kDa.

[0044] Example 3: Performance determination of D-amino acid oxidase (1) Optimal pH and pH stability The optimal pH and pH stability of the wild-type crude enzyme were determined: Optimal pH: The enzyme activity of wild-type crude enzyme was determined in phosphate buffers with different pH values ​​(6.5, 7.0, 7.5, 8.0, 8.5), where the phosphate buffers with different pH values ​​were adjusted with ammonia and phosphate.

[0045] (2) Optimal temperature and temperature stability The optimal temperature and temperature stability of the wild-type crude enzyme were determined: Optimal temperature: The enzyme activity of wild-type crude enzyme was determined under different temperature conditions (25℃, 30℃, 35℃, 40℃).

[0046] The results showed that the D-amino acid oxidase exhibited the highest enzyme activity at 30℃ and pH 8.0 (phosphate buffer). Figure 2 ).

[0047] Similarly, the optimal pH and optimal temperature of the mutant enzyme were determined, and the results showed that: Table 1. Optimal temperature and pH for wild-type and mutant enzymes.

[0048] (3) Detection of D-glufosinate conversion capacity The total system for D-amino acid oxidase mutant enzyme activity assay was 10 mL.

[0049] The crude enzyme solution from Example 2 was brought to a final volume of 10 mL with phosphate buffer (pH 8.0). The solution was preheated in a shaker at 30°C and 200 rpm. D,L-glufosinate-ammonium (30 g / L) and catalase (4 U / mL) were quickly added. The reaction was carried out at 30°C and 200 rpm for 3 hours. A 1 mL sample was then taken, and 10 μL of 6 M HCl was quickly added and mixed thoroughly to terminate the reaction. The concentration of PPO in the reaction solution was determined using HPLC.

[0050] The results are as follows Figure 3 As shown, the enzyme activity of the mutant A51V was increased by 29% compared to the wild type. This indicates that a D-amino acid oxidase with enhanced enzyme activity was successfully obtained through mutation site design.

[0051] Table 2 Enzyme activities of wild-type and mutant enzymes

[0052] Example 4: Recombinant Escherichia coli E.Coli BL21 / pET-28a-Daao A51V -Cat construction Synthesize the D-amino acid oxidase gene Daao containing the mutation from Example 1 A51V (Amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.3) and derived from Bacillus thermophilus ( Geobacillus sp. The gene fragment of the catalase gene Cat (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6) of B1 was then ligated to the EcoliRI and BsrGI restriction sites, and the BseRI and HindIII restriction sites of the pET-28a vector, respectively, to obtain the recombinant vector pET-28a-Daao A51V -Cat. The recombinant plasmid pET-28a-Daao A51V -Cat transformation of E. coli E.Coli BL21 yielded recombinant Escherichia coli. E.Coli BL21 / pET-28a-Daao A51V -Cat.

[0053] D-amino acid oxidase gene DAAO A51V The amino acid sequence (SEQ ID NO. 2): MHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWVGATWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAK SIAGIDDQAAEPIRGQTVLVKSPCKRCTTDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSQAGYQQSWGAAEDVAQLVDEAFQRYHGAARESKL D-amino acid oxidase gene DAAO A51V The nucleotide sequence (SEQ ID NO. 3): The amino acid sequence of the catalase gene (SEQ ID NO. 4): MADTKKLTTSWGAPVGDNQNSITAGNPGPTLIQDVHLIEKLAHFNRERVPERVVHAKGAGAHGYFEVTNDMSKYTKAKVFNGVGKRTPVFVRFSTVAGELGSADTVRDPRGFAVKFYTEEGN YDIVGNNTPIFFIRDAIKFPDFIHTQKRDPRTHLKNPTAMWDFWSLSPESLHQVTYLFGDRGIPLTYRHMNGYGSHTFKWVNEKGEAVWVKYHFKTNQGVKNMDPELAVKIAGENPDYHTED LYNAIEKGDYPSWTLYVQIMPLEDAKTYRFNPFDVTKVWSHKDYPLIEVGRMVLNRNPENYFAEVEQATFSPGNLVPGVEPSPDKMLQARLFAYADAHRYRVGVNHNLLPINRPRVEVNNYQ RDGFMRFDNNGGGSVNYEPNSFGGPTEVSEHKTTPFPVSGMAESVPYDDDDHYTQAGDLYRLMSEEEKARLVKNIVESLKQVTKEEIKLRQIRHFYKADPDYGRRVAEGLGLQVPDDVITNA The nucleotide sequence of the catalase gene (SEQ ID NO. 5): Example 5: Conversion of Escherichia coli PPO-producing bacteria in a 5 L bioreactor 1. Preparation of crude enzyme solution The recombinant Escherichia coli constructed in Example 2 E.Coli BL21 / pET-28a-Daao A51V -Cat was inoculated into 10 mL LB liquid medium and cultured at 37°C and 200 rpm for 12 h. Then, it was transferred to 100 mL TY fermentation medium at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm for 12 h. The seed culture was then transferred to a 5 L bioreactor containing 1.9 L TY fermentation medium at a 5% (v / v) inoculation rate. The pH was maintained at 7.2 using 50% NH3•H2O (v / v), and the dissolved oxygen level was maintained at approximately 30% by adjusting the stirring speed and aeration rate. After culturing at 37°C for 2 h, fed medium was added at a flow rate of 36 mL / h. After another 2 h of culture, IPTG was added to a final concentration of 5 mM, and the temperature was lowered to 25°C for 20 h of induction. When the cell OD in the bioreactor... 600 When the concentration reaches 80°C, the culture is complete. Collect the bacterial culture, wash the cells with phosphate buffer, centrifuge, collect the cells, and sonicate them: 400 W for 1 second, pause for 3 seconds, for 15 minutes. After sonication, centrifuge at 4°C and 8,000 rpm for 20 minutes, collect the supernatant, and obtain the crude enzyme solution.

[0054] 2. Enzymatic catalysis of racemic D-glufosinate All the crude enzyme solution collected in step 1 was put into a 5 L bioreactor, preheated to 30℃ and 200 rpm, with an aeration rate of 10 NL / min, and 60 g of D,L-glufosinate was added at 0 h and 6 h respectively.

[0055] The results are as follows Figure 4 As shown, at 6 h, the substrate added at 0 h reacted almost completely. When 60 g of DL-glufosinate was added again, the concentration of D-glufosinate decreased rapidly within 6-14 h. At 14.5 h, the concentration of D-glufosinate was 2.9 g / L. At 18 h, no D-glufosinate was detected in the sample diluted 2000 times.

[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A D-amino acid oxidase mutant, characterized in that, The D-amino acid oxidase mutant is based on the D-amino acid oxidase shown in SEQ ID NO.1, with alanine at position 51 mutated to valine.

2. A gene encoding the D-amino acid oxidase mutant of claim 1, or a recombinant vector carrying the gene.

3. A cell expressing the mutant of claim 1, or a recombinant cell carrying the gene or recombinant vector of claim 2; Preferably, the recombinant cells are bacteria or fungi as host cells; more preferably, the bacteria are selected from Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum, and the fungi are yeast.

4. A recombinant enzyme catalyst containing the D-amino acid oxidase mutant of claim 1, characterized in that, It can be any of the following forms: (1) Culturing the recombinant cells of claim 3, and isolating cells containing the mutant; (2) Culturing the recombinant cells of claim 3, separating and lysing the cells to obtain a cell lysate containing the mutant; (3) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate.

5. A method for improving the catalytic performance of D-amino acid oxidase, characterized in that, The D-amino acid oxidase mutant is based on the D-amino acid oxidase shown in SEQ ID NO.1, with alanine at position 51 mutated to valine.

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the D-amino acid oxidase mutant of claim 1; Preferably, the genetically engineered bacteria also express at least one coenzyme associated with the production of 2-oxo-4-[(hydroxy)(-methyl)phosphine]butyric acid; More preferably, the coenzyme includes catalase; Preferably, the host of the genetically engineered bacteria is Escherichia coli BL21(DE3).

7. A method for resolving racemic D,L-glufosinate, characterized in that, Using D,L-glufosinate as a substrate, and employing the recombinant cells of claim 3, the recombinase catalyst of claim 4, or the genetically engineered bacteria of claim 6 as catalysts, racemic D,L-glufosinate is cleaved to generate the intermediate PPO.

8. The method according to claim 7, characterized in that, The catalytic system also includes catalase.

9. The method according to claim 7, characterized in that, The catalytic temperature is 25~40℃, and the pH is 7~8.

10. The use of the mutant of claim 1, the gene or recombinant vector of claim 2, the recombinant cell of claim 3, the recombinase catalyst of claim 4, the genetically engineered bacteria of claim 6, or the method of claim 7 or 8 in any of the following: (1) Preparation of L-glufosinate; (2) Preparation of intermediate PPO.

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