Recombinant D-amino acid oxidase mutant, coding gene and application thereof

Abstract

The invention discloses a recombinant D-amino acid oxidase mutant, a coding gene and application thereof, and belongs to the technical field of enzyme engineering. A D-amino acid oxidase mutant is constructed, the catalytic activity of enzyme is greatly improved, and the specific enzyme activity can be improved to 54.32 U / g. The constructed mutant is used for catalyzing D / L-glufosinate-ammonium, under the participation of glutamate dehydrogenase and ammonium formate dehydrogenase, a catalytic reaction can be carried out under the 1M substrate concentration, the catalytic conversion rate is 99%, the yield is 97%, and the e.e. Value is gt; and the effect is 99.9%.

Description

Technical Field

[0001] The present invention relates to a recombinant D - amino acid oxidase mutant, a coding gene thereof and applications thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] L - glufosinate is a herbicide widely used at present, especially important for weed control in genetically modified crops. At present, the synthesis methods of L - glufosinate include chemical methods and biocatalytic methods. The chemical methods have complex processes and high requirements for equipment. Therefore, the biocatalytic method is more economical and environmentally friendly. The mainstream biocatalytic method uses D / L - glufosinate as the substrate. Among them, D - glufosinate is oxidized to generate the intermediate PPO, and then PPO is reductively aminated to obtain L - glufosinate. The theoretical yield of this catalytic method can reach 100%. At present, the technical route of the CN113969269 patent is the same as that of this patent, that is, D - glufosinate is oxidized and reductively aminated to change the configuration to obtain L - glufosinate, but its substrate concentration, conversion rate and space - time yield are low. Summary of the Invention

[0003] The present invention aims to provide a D - amino acid oxidase mutant with high activity, high substrate conversion rate and high selectivity and its application in the catalytic preparation of L - glufosinate.

[0004] The present invention provides a D - amino acid oxidase mutant which has one or more mutations of I15E, N54G, N54A, T56N, P286F, A340L on the basis of the parent.

[0005] In one embodiment, the amino acid sequence of the parent is as shown in Genbank accession number: GAA6022982.1.

[0006] In one embodiment, the mutant is obtained by mutating isoleucine at position 15 to glutamic acid on the basis of the parent, and the obtained mutant is named I15E.

[0007] In one embodiment, the mutant is obtained by mutating asparagine at position 54 to glycine on the basis of the parent, and the obtained mutant is named N54G.

[0008] In one embodiment, the mutant is obtained by mutating asparagine at position 54 to alanine on the basis of the parent, and the obtained mutant is named N54A.

[0009] In one embodiment, the mutant is obtained by mutating threonine at position 56 to asparagine on the basis of the parent, and the obtained mutant is named T56N.

[0010] In one embodiment, the mutant is obtained by mutating proline at position 286 to phenylalanine on the basis of the parent, and the obtained mutant is named P286F.

[0011] In one embodiment, the mutant is obtained by mutating alanine at position 340 to leucine on the basis of the parent, and the obtained mutant is named A340L.

[0012] In one embodiment, the mutation is to mutate isoleucine at position 15 to glutamic acid, asparagine at position 54 to glycine, threonine at position 56 to asparagine, proline at position 286 to phenylalanine, and alanine at position 340 to leucine. The obtained mutant is named I15E / N54G / T56N / P286F / A340L.

[0013] In one embodiment, the mutation is to mutate isoleucine at position 15 to glutamic acid, asparagine at position 54 to alanine, threonine at position 56 to asparagine, proline at position 286 to phenylalanine, and alanine at position 340 to leucine. The obtained mutant is named I15E / N54A / T56N / P286F / A340L.

[0014] In one embodiment, the amino acid sequence of the mutant is as shown in SEQ ID NO.1.

[0015] The present invention also provides an enzyme preparation containing the D - amino acid oxidase mutant.

[0016] The present invention also provides a gene encoding the mutant.

[0017] In one embodiment, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0018] The present invention also provides a recombinant Escherichia coli expressing the mutant.

[0019] In one embodiment, the recombinant Escherichia coli BL21(DE3) is used as the host and pET - 28a is used as the vector to express the mutant.

[0020] The present invention also provides a cell catalyst containing the recombinant Escherichia coli live cells.

[0021] In one embodiment, the cell catalyst is the recombinant Escherichia coli

[0022] In one embodiment, the cell catalyst is obtained by culturing the recombinant Escherichia coli in LB medium for a period of time and then transferring it to TB medium for continued culturing until OD 600= 0.4 - 0.8, induced and cultured with IPTG at a final concentration of 0.05 - 0.2 mM, and the bacterial cells were collected by centrifugation.

[0023] In one embodiment, the induction is carried out at 20 - 25 °C for 16 - 24 h.

[0024] The present invention also provides a method for improving the catalytic activity of D - amino acid oxidase, which is based on the D - amino acid oxidase parent, and mutates one or more amino acids at positions 15, 54, 56, 286, and 340.

[0025] In one embodiment, the method performs the following one or more mutations on the parent:

[0026] (a) Mutate isoleucine at position 15 to glutamic acid;

[0027] (b) Mutate asparagine at position 54 to glycine or alanine;

[0028] (c) Mutate threonine at position 56 to asparagine;

[0029] (d) Mutate proline at position 286 to phenylalanine;

[0030] (e) Mutate alanine at position 340 to leucine.

[0031] In one embodiment, the amino acid sequence of the D - amino acid oxidase parent is as shown in Genbank accession number: GAA6022982.1.

[0032] The present invention also provides a method for catalytically preparing L - glufosinate, which uses D / L - glufosinate as a substrate, and uses the mutant or the cell catalyst to catalytically prepare L - glufosinate in the presence of glutamate dehydrogenase, formate dehydrogenase, and catalase.

[0033] In one embodiment, the glutamate dehydrogenase has the amino acid sequence shown in NCBI reference sequence: WP_274074527.1; the formate dehydrogenase has the amino acid sequence shown in NCBI reference sequence: WP_064248942.1.

[0034] In one embodiment, the concentration of the substrate ≥ 198 g / L.

[0035] In one embodiment, the addition amount of the mutant ≥ 2 g / L.

[0036] In one embodiment, the addition amount of the cell catalyst ≥ 6 g / L.

[0037] In one embodiment, the reaction is carried out at 25 - 30 °C for 10 - 20 h.

[0038] The present invention also provides the use of the D - amino acid oxidase mutant, or the recombinant Escherichia coli, or the method in the preparation of L - glufosinate or herbicides containing L - glufosinate.

[0039] Beneficial effects:

[0040] 1. The present invention constructs a D - amino acid oxidase mutant, which solves the problem that the catalytic pocket of the wild - type enzyme is closed and the substrate cannot enter the catalytic pocket for reaction, so that the closed pocket of the constructed mutant is opened, greatly improving the catalytic activity. The specific enzyme activity can be increased to 54.32 U / g; compared with the enzymes in the prior art, the substrate concentration can be catalyzed up to 198 g / L within the same time.

[0041] 2. The present invention provides the use of the D - amino acid oxidase mutant in catalyzing the preparation of L - glufosinate from a D / L - glufosinate mixture or D - glufosinate. In the presence of glutamate dehydrogenase and formate dehydrogenase, the mutant constructed by the present invention can catalyze at a substrate concentration of 1 M, achieving a catalytic conversion rate of 99%, a yield of 97%, and an e.e. value > 99.9%. Description of the drawings

[0042] Figure 1 It is a schematic diagram of the catalytic reaction of D - amino acid oxidase. Detailed implementation manners

[0043] (I) Determination method:

[0044] Determination of D - amino acid oxidase: Prepare a 30 - mL catalytic system containing 0.2 g of pure D - amino acid oxidase, 0.15 g of catalase, and 6 g of glufosinate powder. Adjust the pH to 8.0 with ammonia water, and then react at 30 °C, 250 r / min, and 3.0 vvm for 2 h to determine the content and configuration of glufosinate in the system.

[0045] The definition of enzyme activity unit (U) is: at the optimal temperature and optimal pH, the amount of enzyme required to catalyze 1 μmol of D - PPT to the product per minute is one activity unit, that is, 1 U.

[0046] Determination of glufosinate content: Dilute the above sample with pure water by an appropriate multiple and then filter it through a 0.22 μm filter membrane to obtain the test sample. The detection conditions are as follows: The chromatographic column is EC-C18 100×4.6 mm, 4 μm; the liquid phase is Agilent Poroshell120, the mobile phase is a mixture of 0.1% tetrabutylammonium hydrogen sulfate - 50 mM ammonium dihydrogen phosphate (pH = 3.8) and methanol in a volume ratio of 88:12, the injection volume is 5 μL, the column temperature is set at 35°C, the running time is set at 10 min, and the detection wavelength is 195 nm. The detection results are used for calculating the substrate conversion rate and enzyme activity.

[0047] Determination of glufosinate configuration: The product configuration determination requires liquid phase analysis after filtering through a 0.22 μm filter membrane. The chromatographic column is micropuliteGoldC18 150*4.6 mm 5 μm from Micro-Pure Biotechnology Co., Ltd., the mobile phase is a methanol solution of 50 mM sodium acetate (pH 6.0) and pure methanol, the injection volume is 5 μL, the column temperature is set at 40°C, the running time is set at 10 min, and the detection wavelength is 338 nm.

[0048] (II) Preparation of enzymes:

[0049] Glutamate dehydrogenase (Genbank: WP_274074527.1): Synthesize the gene sequence encoding glutamate dehydrogenase (shown as SEQ ID NO.3), use pET-28a as the vector, and Escherichia coli BL21(DE3) as the host to construct a recombinant bacterium expressing glutamate dehydrogenase. Culture the constructed recombinant bacterium at 37°C for 2 h, and then culture it at 25°C under the condition of 0.1 mM IPTG for about 18 h. Collect the fermentation broth and perform cell disruption to obtain glutamate dehydrogenase.

[0050] Ammonium formate dehydrogenase (Genbank: WP_064248942.1): Synthesize the gene sequence encoding ammonium formate dehydrogenase (shown as SEQ ID NO.4), use pET-28a as the vector, and Escherichia coli BL21(DE3) as the host to construct a recombinant bacterium expressing ammonium formate dehydrogenase. Culture the constructed recombinant bacterium at 37°C for 2 h, and then culture it at 25°C under the condition of 0.1 mM IPTG for about 18 h. Collect the fermentation broth and perform cell disruption to obtain ammonium formate dehydrogenase.

[0051] Protein purification: The fermented broth was centrifuged to obtain the bacterial cells. 8 g of the above-mentioned bacterial cells were weighed and added to 100 mL of Binding buffer (500 mM NaCl, 20 mM NaH2PO4, pH 7.5), and stirred in an ice bath until completely dissolved. Ultrasonic disruption was carried out for 30 min under the conditions of a power ratio of 40%, ultrasonic / interval: 3 / 5 s. Subsequently, the disrupted solution was centrifuged, and the obtained supernatant was filtered through a 0.22 μm filter membrane to remove impurities. Protein purification was performed using an AKTA Purifier 10 instrument. The Binding buffer was used to equilibrate the nickel column Ni-NTA 6FF at a flow rate of 5 mL / min. After stabilization, the sample was loaded onto the nickel column at a flow rate of 1 mL / min. After stabilization, the contaminant proteins were eluted with 5 column volumes of Washing buffer (500 mM NaCl, 20 mM NaH2PO4, 20 mM imidazole, flow rate 1 mL / min). After the elution was completed, the target protein was eluted and collected with 5 column volumes of Elution buffer (500 mM NaCl, 20 mM NaH2PO4, 300 mM imidazole, pH 7.5, flow rate 1 mL / min). After verification by SDS-PAGE gel electrophoresis, the eluate containing the target protein was filtered and concentrated using a 10-30 kDa ultrafiltration tube, and the imidazole in the enzyme solution was removed by washing with potassium phosphate buffer (50 mM, pH 7.0). The protein concentration in the pure enzyme solution was determined using a BCA kit (Beyotime), and after adding 20% glycerol, it was stored in a -80 °C refrigerator.

[0052] Example 1

[0053] Using the D-amino acid oxidase derived from Rhodotorula taiwanensis as a gene probe, six D-amino acid oxidases (Genbank accession numbers are ALM22238.1, TNY23393.1, KAK4701668.1, XP_052943252.1, ORY22187.1, and GAA6022982.1, respectively) derived from Sporobolomyces roseus, Rhodotorula diobovata, Phenoliferia sp. Uapishka_3, Dioszegia hungarica, Naematelia encephala, and Rhodotorula sphaerocarpa were screened out in the NCBI database. After codon optimization for Escherichia coli, the sequences shown in SEQ ID NO.5 to SEQ ID NO.10 were obtained respectively. The codon-optimized D-amino acid oxidase genes were respectively integrated into the pET-28a plasmid and heterologously expressed in Escherichia coli BL21(DE3). The specific steps are as follows:

[0054] The recombinant Escherichia coli was cultured in LB medium at 37 °C for 12 h, and then transferred to TB medium at an inoculation amount of 2%, and cultured at 37 °C until OD 600 600 = 0.6. IPTG was added to a final concentration of 0.1 mM, and finally the cells in the fermentation broth were collected by centrifugation after culturing at 25 °C for 20 h. After protein purification, the catalytic ability of glufosinate was detected. The results showed (Table 1) that the catalytic activity of D-amino acid oxidase derived from Rhodotorula sphaerocarpa was 0.06 ± 0.02 U / g as verified by activity.

[0055] Table 1 Comparison of catalytic abilities of different D-amino acid oxidases

[0056]

[0057] Example 2 Construction and screening of mutants

[0058] Using D-amino acid oxidase (DAAO6) derived from Rhodotorulasphaerocarpa as the parental enzyme (the amino acid sequence is shown as Genbank accession number: GAA6022982.1), protein engineering was carried out to improve the activity and catalytic efficiency of DAAO6.

[0059] DAAO6 was subjected to homology modeling using 1c0k (PDBid) as a template to obtain its protein structure. Subsequently, the active pocket residues of DAAO6 were analyzed by molecular docking with FAD. Finally, through sequence alignment and analysis of the flexibility of the loop at the subunit contact surface, a total of 26 saturation mutation sites were selected near its active pocket: G13, V14, I15, S48, P49, W50, G52, A53, N54, F55, T56, T163, L181, G182, K184, L239, V240, L284, R285, P286, A287, S338, S339, A340, G341, Y342.

[0060] Synthesize the gene sequence of DAAO6 containing the nucleotide sequence as shown in SEQ ID NO.10, and ligate the gene sequence into the plasmid pET-28a. Design primers and introduce mutations at the above sites using PCR technology. The specific method is as follows: The reaction system is 25 μL of PrimeStarMax enzyme, 2 μL of each of the upstream and downstream mixed primers, 1 μL of DAAO6 plasmid, and make up to 50 μL with pure water. The PCR reaction conditions are denaturation at 98°C for 5 min, then denaturation at 98°C for 30 s, annealing at 58°C for 15 s, extension at 72°C for 1 min 10 s. After 30 cycles, extend at 72°C for 10 min. After the amplification, a fragment product of about 6300 bp is obtained. The product is digested with DpnⅠ enzyme and then transformed into Escherichia coli BL21(DE3) competent cells. After coating, it is cultured overnight at 37°C to obtain a mutant library.

[0061] Screen the mutant library using high-throughput screening: Culture the bacteria and centrifuge to obtain cells according to the method of Example 1. Take 0.2 g of cells and add 100 μL of lysozyme solution to break them. Centrifuge and take the supernatant for determination. The system includes 0.016 g / mL of D / L-PPT, 0.005 g / mL of 4-aminoantipyrine, 0.025 g / mL of tetrabutylammonium bromide, 0.006 g / mL of horseradish peroxidase, and 0.25 mL / mL of DAAO lysate supernatant. The system reacts at 25°C for 10 min, and then measure the absorbance at 510 nm.

[0062] Six single-point mutants DAAO7 - DAAO12 with significantly improved activities were screened out from 2184 single colonies. Combine these single-point mutants to obtain combined mutants DAAO13 - DAAO14, and measure the activities of each mutant. The results are shown in Table 2. The specific enzyme activities of mutants DAAO7 - DAAO14 are significantly higher than those of the parent (DAAO6).

[0063] Table 2 Comparison of the catalytic abilities of different D-amino acid oxidase mutants

[0064] Mutant Mutation site Specific activity of mutant (U / g) DAAO7 I15E 17.65±0.97 DAAO8 N54G 11.61±2.29 DAAO9 N54A 13.22±3.27 DAAO10 T56N 23.56±1.89 DAAO11 P286F 18.24±2.48 DAAO12 A340L 18.07±3.55 DAAO13 I15E / N54G / T56N / P286F / A340L 42.98±2.89 DAAO14 I15E / N54A / T56N / P286F / A340L 54.32±1.45

[0065] Example 3 Application of Mutant DAAO14

[0066] Express the recombinant strain DAAO14 according to the method of Example 1, and collect the wet bacterial cells of the recombinant bacteria by centrifugation. Using the wet bacterial cells as a catalyst, D / L-glufosinate as a substrate, catalase (purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd.) is added to decompose the by-product hydrogen peroxide, and glutamate dehydrogenase (NCBI reference sequence: WP_274074527.1) and formate dehydrogenase (NCBI reference sequence: WP_064248942.1) are added to catalyze the formation of L-glufosinate and coenzyme recycling from PPO, NADPH is added as a coenzyme, and ammonium formate is added for coenzyme recycling and amino donor. A reaction system is constituted with 0.05M Tris-HCl buffer solution at pH 8.0 as the reaction medium for the reaction. The specific process is as follows:

[0067] Prepare a reaction system containing (by final concentration): D / L-glufosinate 198 g / L, catalase 500 U / mL, ammonium formate 0.6 M, NADP 0.1 mM, and then add DAAO14 wet bacterial cells 6.66 g / L (converted to about 2.5 g / L of pure enzyme participating in the reaction), cell lysate expressing glutamate dehydrogenase 7 g / L, and cell lysate expressing formate dehydrogenase 13 g / L by final concentration, and adjust the pH to 8.0 with ammonia water. The reaction temperature is set at 30 °C, the rotation speed is set at 250 r / min, air is introduced (3 vvm), and after catalyzing for 12 - 14 h, the reaction ends, and the L-glufosinate catalytic feed liquid is obtained. The substrate conversion rate reaches 99%, the ee value of the product reaches 99.9%, and the catalytic yield reaches over 97%.

[0068] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A D - amino acid oxidase mutant, characterized in that, Based on the parent, having one or more mutations among I15E, N54G, N54A, T56N, P286F, and A340L.

2. The D-amino acid oxidase mutant according to claim 1, wherein The parent has the amino acid sequence shown by Genbank accession number: GAA6022982.

1.

3. The D - amino acid oxidase mutant according to claim 1 or 2, characterized in that, The mutant, compared with the parent, has at least one of the following mutations: (a) Mutating isoleucine at position 15 to glutamic acid; (b) Mutating asparagine at position 54 to glycine or alanine; (c) Mutating threonine at position 56 to asparagine; (d) Mutating proline at position 286 to phenylalanine; (e) Mutating alanine at position 340 to leucine.

4. A gene encoding the D - amino acid oxidase mutant according to any one of claims 1 to 3.

5. A recombinant Escherichia coli expressing the D - amino acid oxidase mutant according to any one of claims 1 to 3, or the gene according to claim 4.

6. An enzyme preparation containing the D - amino acid oxidase mutant according to any one of claims 1 to 3.

7. A cell catalyst containing the live cells of the recombinant Escherichia coli according to claim 5.

8. A method for improving the catalytic activity of D - amino acid oxidase, characterized in that, Based on the D - amino acid oxidase parent, mutating one or more amino acids at positions 15, 54, 56, 286, and 340; the parent has the amino acid sequence shown by Genbank accession number: GAA6022982.

1.

9. A method for catalytically preparing L-glufosinate, characterized in that, Using D / L - glufosinate as a substrate, in the presence of glutamate dehydrogenase and formate dehydrogenase, catalyzing the preparation of L - glufosinate by using the D - amino acid oxidase mutant according to any one of claims 1 to 3 or the cell catalyst according to claim 7.

10. Use of the D - amino acid oxidase mutant according to any one of claims 1 to 3, or the recombinant Escherichia coli according to claim 5, or the method according to any one of claims 8 to 9 in the preparation of L - glufosinate or a herbicide containing L - glufosinate.

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