Preparation method of xanthine oxidase

Abstract

The invention provides a preparation method of xanthine oxidase, and belongs to the technical field of molecular biology. The invention provides a preparation method of xanthine oxidase, which comprises the following steps: taking pseudomonas as host bacteria, introducing three genes xdhA, xdhB and xdhC into the genome of the host bacteria, completing the expression of xanthine dehydrogenase by a co-expression method, and treating the xanthine dehydrogenase by using a sulfydryl specific modifier to produce the xanthine oxidase. The precursor substance of xanthine oxidase is prepared through fermentation of recombinant pseudomonas, efficient expression in pseudomonas is achieved, the specific activity of an expression product after purification is 100.3 U / mg, and the expression product can be used for industrial production application after being treated by a sulfydryl specific modifier.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for preparing xanthine oxidase. Background Art

[0002] Xanthine oxidoreductase (XOR) is an oxidoreductase that exists in two different forms: xanthine oxidase (XOD, EC 1.17.3.2) and xanthine dehydrogenase (XDH, EC 1.17.1.4). Xanthine oxidase mainly catalyzes the oxidation of hypoxanthine to xanthine, which is then further oxidized to produce uric acid.

[0003] At present, the XOR widely used in the market is XOD, which is mainly extracted from milk. Milk xanthine oxidase is the main commercial product on the market and the earliest enzyme to be patented on the market. Other commercial XORs mainly include E.cloacae XOD produced by Kikkoman Company and A.luteus XOD provided by Toyobo Company. XOD has high commercial value and is widely used in clinical diagnosis, drug synthesis, food and environmental protection. However, with the continuous expansion of application fields, commercial XOD still faces the problems of few enzyme types, low enzyme catalytic activity and low production efficiency. Therefore, the research on large-scale and low-cost production methods of XOD can create huge economic and social benefits for our country. Summary of the invention

[0004] The present invention provides a method for preparing xanthine oxidase. By expressing the enzyme in a natural host (Pseudomonas), the modification and function of the enzyme are consistent with the natural state, the expression in the natural environment is more stable and not easy to degrade, and the protein has little cytotoxicity even if expressed in large quantities, thereby achieving efficient expression of xanthine oxidase in Pseudomonas, and the purified xanthine oxidase has high specific activity.

[0005] In order to solve the above technical problems, the present invention provides a method for preparing xanthine oxidase, comprising introducing three genes, xdhA, xdhB and xdhC, into the genome of Pseudomonas, and co-expressing them in the Pseudomonas, wherein the expression product contains xanthine dehydrogenase;

[0006] The xanthine dehydrogenase is treated with a thiol-specific modifier to obtain xanthine oxidase.

[0007] In a preferred embodiment of the present invention, the sources of the three genes xdhA, xdhB and xdhC include Pseudomonas aeruginosa PAO1.

[0008] In a preferred embodiment of the present invention, the nucleotide sequences of the three genes xdhA, xdhB and xdhC derived from Pseudomonas aeruginosa PAO1 are shown in SEQ ID No.1 to SEQ ID No.3.

[0009] In a preferred embodiment of the present invention, when the three genes xdhA, xdhB and xdhC are introduced into the genome of the Pseudomonas, the amplified fragment containing the xdhA and xdhB genes is first inserted into an expression vector to construct a first expression vector; the amplified xdhC gene is inserted into the first expression vector to construct a second expression vector, and the second expression vector is used to transform Pseudomonas aeruginosa PAO1 to construct a co-expression recombinant Pseudomonas.

[0010] In a preferred embodiment of the present invention, the amplification primers for the fragment containing the xdhA and xdhB genes include xdhAB-F and xdhAB-R, the nucleotide sequence of xdhAB-F is shown in SEQ ID No.4, and the nucleotide sequence of xdhAB-R is shown in SEQ ID No.5.

[0011] In a preferred embodiment of the present invention, when the expression vector is pRGPDuo2, the fragment containing the xdhA and xdhB genes is inserted into the Pst I and BamHI sites of the vector to construct a recombinant plasmid pRGPDuo2-xdhAB.

[0012] In a preferred embodiment of the present invention, the amplification primers for the xdhC gene include xdhC-F and xdhC-R, the nucleotide sequence of xdhC-F is shown in SEQ ID No.6, and the nucleotide sequence of xdhC-R is shown in SEQ ID No.7.

[0013] In a preferred embodiment of the present invention, the procedure for amplifying the fragment containing the xdhA and xdhB genes includes: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 4 min, 34 cycles; and extension at 72°C for another 5 min.

[0014] The procedure for amplifying the fragment of the xdhC gene included: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 1 min, 34 cycles; and extension at 72°C for another 5 min.

[0015] In a preferred embodiment of the present invention, the xdhC gene is inserted into two restriction sites, BgIII and Bmt I, of the vector pRGPDuo2-xdhAB.

[0016] In a preferred embodiment of the present invention, the thiol-specific modifier comprises 5,5'-dithiobis(2-nitrobenzoic acid).

[0017] Beneficial effects: The present invention provides a method for preparing xanthine oxidase, using Pseudomonas as a host bacterium, introducing three genes xdhA, xdhB and xdhC into the genome of the host bacterium, and completing the expression of xanthine dehydrogenase by a co-expression method, and then treating the xanthine dehydrogenase with a thiol-specific modifier to produce xanthine oxidase. The present invention prepares a precursor substance of xanthine oxidase by fermentation of recombinant Pseudomonas, realizes efficient expression in Pseudomonas, and the specific activity of the expression product after purification is 100.3U / mg, and can be used for industrial production and application after being treated with a thiol-specific modifier. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1 is the result of PCR amplification of xdhAB and xdhC genes;

[0019] Figure 2 This is the result of colony PCR identification of pRGPDuo2-xdhAB;

[0020] Figure 3 This is the result of colony PCR identification of pRGPDuo2-xdhABC;

[0021] Figure 4 is the electropherogram of the extracted Pseudomonas aeruginosa PAO1 genome;

[0022] Figure 5 This is a concentration standard curve graph made with BSA;

[0023] Figure 6 The figure is the electrophoresis diagram of the purified xanthine dehydrogenase expressed by the recombinant bacteria; M is the protein molecule marker; Lane 1 is the higher concentration protein No. 3 collected after purification. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing xanthine oxidase, comprising introducing three genes, xdhA, xdhB and xdhC, into the genome of Pseudomonas, and co-expressing them in the Pseudomonas, wherein the expression product contains xanthine dehydrogenase;

[0025] The xanthine dehydrogenase is treated with a thiol-specific modifier to obtain xanthine oxidase.

[0026] In a preferred embodiment of the present invention, the sources of the three genes xdhA, xdhB and xdhC include Pseudomonas aeruginosa PAO1, and the nucleotide sequences of the three genes xdhA, xdhB and xdhC derived from Pseudomonas aeruginosa PAO1 are shown in SEQ ID No.1 to SEQ ID No.3, respectively.

[0027] In one embodiment of the present invention, three genes xdhA, xdhB and xdhC are amplified from Pseudomonas aeruginosa PAO1 by PCR amplification. Among them, xdhA (Gene ID: 879268) is a coding sequence for the small subunit of xanthine dehydrogenase, with a size of about 48.5KDa; xdhB (Gene ID: 879250) is a coding sequence for the large subunit of xanthine dehydrogenase, with a size of about 87.6KDa; xdhC is a chaperone protein for xanthine dehydrogenase, with a size of about 29.5KDa. When the present invention amplifies the three genes, the large and small subunits of xanthine dehydrogenase are fused and expressed (xdhAB), and the molecular chaperone xdhC (Gene ID: 883006) is additionally expressed to mediate the correct folding of xanthine dehydrogenase, thereby increasing the yield of xanthine dehydrogenase.

[0028] In one embodiment of the present invention, the xdhAB fragment is amplified using primers xdhAB-F and xdhAB-R, the nucleotide sequence of the xdhAB-F is shown in SEQ ID No.4, the nucleotide sequence of the xdhAB-R is shown in SEQ ID No.5, and the amplification procedure includes: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 62°C for 15 seconds, extension at 72°C for 4 minutes, 34 cycles; and extension at 72°C for another 5 minutes.

[0029] Upstream primer xdhAB-F (SEQ ID No.4):

[0030] 5′-gccaagcttgcatgcctgcagATGATCAGGTTCCTGCTCAACC-3′;

[0031] Downstream primer xdhAB-R (SEQ ID No.5):

[0032] 5′-gagctcggtacccggggatccTCAGGCCGGTTCGACCGC-3′.

[0033] The present invention utilizes a Pseudomonas aeruginosa expression vector, inserts the amplified xdhAB gene into the expression vector, and in one embodiment, pRGPDuo2 is used as an expression vector, and the xdhAB gene is inserted into the Pst I and BamH I sites of pRGPDuo2 using a double enzyme digestion method to construct a recombinant plasmid pRGPDuo2-xdhAB. The pRGPDuo2 plasmid of the present invention is purchased from Baosai Biotechnology and is a dual-promoter high expression vector for Pseudomonas.

[0034] The present invention utilizes a PCR amplification method and primers xdhC-F and xdhC-R to amplify the xdhC gene from Pseudomonas aeruginosa PAO1, wherein the nucleotide sequence of the xdhC-F is shown in SEQ ID No.6, and the nucleotide sequence of the xdhC-R is shown in SEQ ID No.7, and the amplification procedure comprises: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 62°C for 15 seconds, extension at 72°C for 1 minute, and 34 cycles; and extension at 72°C for another 5 minutes.

[0035] Upstream primer xdhC-F (SEQ ID No.6):

[0036] 5′-agaaaagaattcaaaagatctATGAACTGGATCAGCGCCC-3′;

[0037] Downstream primer xdhC-R (SEQ ID No.7):

[0038] 5′-aatttaaatcctagggctagcTCAGGATTCGTGGGCGCG-3′.

[0039] In the present invention, the amplified xdhC gene is inserted into the recombinant plasmid pRGPDuo2-xdhAB. In one embodiment, the insertion sites are BgIII and Bmt I, and the recombinant expression plasmid pRGPDuo2-xdhABC is constructed.

[0040] The present invention utilizes the recombinant expression plasmid pRGPDuo2-xdhABC to electroporate Pseudomonas aeruginosa PAO1 to construct a co-expression recombinant Pseudomonas. The present invention does not specifically limit the transformation method, and the transformation can be performed using conventional methods in the art.

[0041] The invention cultivates the recombinant Pseudomonas obtained after transformation, and the culture medium used is LB culture medium, and the culture medium is 37°C, 220rpm, and 48h, and 0.2mM inducer IPTG is added. The recombinant Pseudomonas cultured in the above method contains xanthine dehydrogenase.

[0042] The present invention centrifuges the fermentation liquid, collects the precipitate, suspends the bacteria in a phosphate buffer (50mM, pH7.5), ultrasonically disrupts at 4°C for 25min, ultrasonicates for 3s, rests for 3s, centrifuges the cell disrupted liquid at 4°C, collects the supernatant, and the supernatant is the crude enzyme liquid.

[0043] The present invention purifies the crude enzyme solution. In one embodiment, a nickel column affinity column is equilibrated with a 20mmol / L pH 7.5 phosphate buffer, the obtained crude enzyme solution is filtered with a 0.22μm water system microporous filter membrane, the filtrate is loaded on the nickel column affinity column, and the impurity protein is washed with a 20mmol / L pH 7.5 phosphate buffer. The volume of each elution is at least 10 column volumes. The phosphate buffer of 10, 50, 100, 200, 300, and 400mM imidazole is used for stage elution at a flow rate of 1mL / min. The eluate of each stage is collected, and the sample is detected by SDS-PAGE. The collected eluate is the target recombinant enzyme.

[0044] The recombinant enzyme of the present invention is xanthine dehydrogenase after experiments and needs to be treated with a thiol-specific modifier. The thiol-specific modifier includes 5,5'-dithiobis(2-nitrobenzoic acid). After the treatment, the xanthine oxidase can be obtained.

[0045] In order to further illustrate the present invention, a method for preparing xanthine oxidase provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the reagents used in the examples of the present invention are conventional reagents in the art and can be purchased. For example, the plasmid DNA extraction kit, DNA gel recovery kit, Taq DNA polymerase and various restriction endonucleases used in the examples are all purchased from Thermo Company. The seamless cloning reagent is from ABclonal, the genome extraction kit is from Yisheng, and the remaining reagents are all domestically produced or imported analytical grade.

[0047] The LB (m / m) medium used in the present invention includes 0.5% yeast powder, 1% peptone and 1% sodium chloride; the final concentration of kanamycin is 50 μg / mL;

[0048] Solid LB medium: Add 2% agar to liquid LB medium to make solid agar plate medium.

[0049] Example 1

[0050] Cloning of Xanthine Dehydrogenase Gene:

[0051] Pseudomonas aeruginosa PAO1 was streaked on LB solid medium in three lines and cultured in a 37°C incubator for 12 h. A single colony was taken in an LB conical flask and cultured overnight at 37°C and 220 rpm. Pseudomonas aeruginosa PAO1 cells were collected by centrifugation. The genomic DNA of Pseudomonas aeruginosa PAO1 was extracted using the Yisheng genome extraction kit ( Figure 4 ). The extracted genomic DNA was used as a template and primers xdhAB-F / R and xdhC-F / R were used for PCR amplification to obtain the gene sequences of the three subunits of xanthine dehydrogenase, xdhAB and xdhC.

[0052] PAO1 was used as a template and PCR amplification was performed using primers xdhAB-F / R and xdhC-F / R. The agarose gel electrophoresis results of the PCR amplification products were as follows: Figure 1 As shown, the lengths of the amplified xdhAB and xdhC gene products are approximately 4000 bp and 900 bp, respectively, which are basically consistent with the gene lengths of the three subunits of xanthine dehydrogenase of Pseudomonas aeruginosa PAO1 in the NCBI GenBank database. The sequencing results show that the correct genes of the three subunits of xanthine dehydrogenase were obtained.

[0053] Example 2

[0054] Construction of recombinant expression plasmid pRGPDuo2-xdhAB:

[0055] The PCR product xdhAB obtained in Example 1 was recovered by agarose gel electrophoresis, and the pRGPDuo2 vector was linearized by double restriction digestion with Pst I and BamHI. The PCR product and the linearized vector pRGPDuo2 were connected by seamless cloning (purchased from ABclonal 2X MultiF Seamless Assembly Mix), transferred into DH5α, and coated on LB solid plates containing kanamycin. Colony PCR was performed to identify transformants. The results are shown in Figure 2 As shown, the size of the xdhAB gene is about 4000 bp, and the bands after colony PCR can be seen, which is consistent with the size of the gene xdhAB. Therefore, xdhAB was successfully cloned into the pRGPDuo2 vector.

[0056] The correct transformants were selected and inoculated into liquid LB medium and cultured at 37°C for 12 h. The plasmid was extracted using a plasmid extraction kit and sent to Shanghai Bioengineering for sequencing. The correct recombinant plasmid was named pRGPDuo2-xdhAB.

[0057] Example 3

[0058] Construction of recombinant expression plasmid pRGPDuo2-xdhABC:

[0059] The PCR product xdhC obtained in Example 1 was recovered by agarose gel electrophoresis, and the product was ligated to the BgIII and Bmt I restriction sites of the pRGPDuo2-xdhAB vector, and then transformed into DH5α, and colony PCR was performed to identify the transformants. Figure 3 As shown, the size of the xdhC gene is about 900 bp, and the band after colony PCR can be seen, which is consistent with the size of the gene xdhC. Therefore, xdhC was successfully cloned into the pRGPDuo2-xdhAB vector.

[0060] The correct transformants were selected and inoculated into liquid LB medium and cultured at 37°C for 12 h. The plasmid was extracted using a plasmid extraction kit and sent to Shanghai Bioengineering for sequencing. The correct recombinant plasmid was named pRGPDuo2-xdhABC.

[0061] Example 4

[0062] Expression and purification of recombinant xanthine oxidase:

[0063] The vector pRGPDuo2-xdhABC constructed in Example 3 was electroporated into the competent cells of Pseudomonas aeruginosa, cultured overnight at 37°C, and positive clones were screened. The recombinant bacteria were named PAO1-xdhABC. The recombinant bacteria were cultured overnight in LB medium and amplified in fermentation medium at 1% inoculum for 48 h (with the inducer 0.2 mM IPTG).

[0064] The fermentation broth was centrifuged, the precipitate was collected, the bacteria were suspended in phosphate buffer (50mM, pH7.5), ultrasonically disrupted at 4℃ for 25min, ultrasonicated for 3s, rested for 3s, the cell disruption solution was centrifuged at 4℃, and the supernatant was collected. The supernatant was the crude enzyme solution. The nickel column affinity column was equilibrated with 20mM pH 7.5 phosphate buffer, the obtained crude enzyme solution was filtered with a 0.22μm water system microporous filter membrane, the filtrate was loaded on the nickel column affinity column, and the impurities were washed with 20mM pH 7.5 phosphate buffer. The volume of each elution was at least 10 column volumes, and 10, 50, 100, 200, 300, 400mM imidazole phosphate buffer was used for stage elution, the flow rate was 1mL / min, and the eluent was a phosphate buffer containing different concentrations of imidazole (NaH2PO4 and Na2HPO4) ， Collect the eluate at each stage, detect the sample by SDS-PAGE, and collect the eluate as the target recombinant enzyme. The results are as follows Figure 6 As shown, the collected recombinant enzyme had the highest enzyme activity in 100 mM imidazole eluate, with an enzyme activity of 80.2 U / mL and a specific activity of 100.3 U / mg.

[0065] Example 5

[0066] Enzyme activity detection of recombinant xanthine oxidase:

[0067] The activity of the recombinant enzyme XOD produced in Example 4 was detected by ultraviolet spectrophotometry. Ultraviolet spectrophotometry is mainly based on the fact that xanthine oxidase consumes 1 mol of molecular oxygen and water to produce 1 mol of uric acid and 1 mol of H2O2 for every 1 mol of xanthine oxidized during the reaction process of catalyzing the oxidation of xanthine, and uric acid has a specific absorption peak at 293nm. The uric acid concentration can be determined by measuring the absorbance of the solution at 293nm, thereby calculating the XOD enzyme activity by the change in uric acid concentration. 1U enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of uric acid at 37°C for 1min. The extinction coefficient per millimole of uric acid under this detection condition is 12.5.

[0068] The specific reagents and steps are as follows:

[0069] Reagent preparation:

[0070] Solution A: 100 mM Tris-HCl buffer (pH 7.5);

[0071] Solution B: 25 mM sodium hydroxide solution (take 0.01 g and dissolve in 10 ml H2O);

[0072] Solution C: 10 mM xanthine solution (15.2 mg xanthine is weighed and dissolved in solution B);

[0073] Solution D: 1 mM potassium oxocyanate solution (9.75 mg potassium oxocyanate dissolved in 50 mL H2O);

[0074] Determination steps:

[0075] 1. Take 1.12 mL of solution A, 0.04 mL of solution C, and 0.04 mL of solution D in a centrifuge tube, incubate at 37°C for 5 min, and then perform UV scanning (220 nm ~ 450 nm);

[0076] 2. Add 50 μL of enzyme solution and gently mix by inverting, incubate at 37°C for 3-5 minutes, and then perform UV scanning (220nm-450nm);

[0077] 3. Calculate the enzyme activity based on the change in absorbance of the reaction solution at 293 nm.

[0078] Select No. 3 (100 mM imidazole eluate) with higher activity and centrifuge it in an ultrafiltration centrifuge tube at 4°C, 4000 g for 20 min. The purified enzyme solution is freeze-dried and stored.

[0079] The protein concentration was detected by BSA method. Prepare BSA standard solutions of different concentrations (0.5, 0.25, 0.15, 0.05, 0.025 mg / ml), measure the absorbance at 550 nm at different concentrations, and draw a standard curve, such as Figure 5As shown, the concentration of BSA standard in the range of 0.05-0.5 mg / mL was similar to that of A 550 The standard curve equation is y=1.035x+0.115, R 2 =0.994, which can be used for protein concentration calculation. According to the standard curve, the protein concentration of the purified PAO1-xdhABC sample was calculated to be 0.8 mg / mL.

[0080] The vector pRGPDuo2 used has two replication origins, colE1 for replication in Escherichia coli and pRO1600 for replication in Pseudomonas putida. The two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are controlled by the inducible promoter Ptac or PtetR / tetA, respectively. Compared with traditional vectors with a single cloning site, its expression efficiency is higher, and the selected xdhAB and xdhC are both from Pseudomonas aeruginosa PAO1 itself, and homologous expression in PAO1 can ensure the correct translation and modification of the protein, efficient expression, and further improve the enzyme activity.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing xanthine oxidase, characterized in that: The method comprises introducing three genes, xdhA, xdhB and xdhC, into the genome of Pseudomonas and co-expressing them in the Pseudomonas, wherein the expression product contains xanthine dehydrogenase; The xanthine dehydrogenase is treated with a thiol-specific modifier to obtain xanthine oxidase.

2. The preparation method according to claim 1, characterized in that: The sources of the three genes xdhA, xdhB and xdhC include Pseudomonas aeruginosa PAO1.

3. The preparation method according to claim 2, characterized in that: The nucleotide sequences of the three genes xdhA, xdhB and xdhC derived from Pseudomonas aeruginosa PAO1 are shown in SEQ ID No.1 to SEQ ID No.

3.

4. The preparation method according to claim 2, characterized in that: When the three genes xdhA, xdhB and xdhC are introduced into the genome of the Pseudomonas, the amplified fragment containing the xdhA and xdhB genes is first inserted into an expression vector to construct a first expression vector; the amplified xdhC gene is inserted into the first expression vector to construct a second expression vector, and the second expression vector is used to transform Pseudomonas aeruginosa PAO1 to construct a co-expression recombinant Pseudomonas.

5. The preparation method according to claim 4, characterized in that: The amplification primers for the fragment containing the xdhA and xdhB genes include xdhAB-F and xdhAB-R, the nucleotide sequence of xdhAB-F is shown in SEQ ID No.4, and the nucleotide sequence of xdhAB-R is shown in SEQ ID No.

5.

6. The preparation method according to claim 4 or 5, characterized in that: When the expression vector is pRGPDuo2, the process comprises inserting the fragment containing the xdhA and xdhB genes into the Pst I and BamHI sites of the vector to construct a recombinant plasmid pRGPDuo2-xdhAB.

7. The preparation method according to claim 4, characterized in that: The amplification primers of the xdhC gene include xdhC-F and xdhC-R, the nucleotide sequence of xdhC-F is shown as SEQ ID No.6, and the nucleotide sequence of xdhC-R is shown as SEQ ID No.

7.

8. The preparation method according to claim 5 or 7, characterized in that: The procedure for amplifying the fragment containing the xdhA and xdhB genes included: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 4 min, 34 cycles; and extension at 72°C for another 5 min; The procedure for amplifying the fragment of the xdhC gene included: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 1 min, 34 cycles; and extension at 72°C for another 5 min.

9. The preparation method according to claim 4 or 7, characterized in that: The method comprises inserting the xdhC gene into BgIII and Bmt I of a first expression vector.

10. The preparation method according to claim 1, characterized in that: The thiol-specific modifier includes 5,5'-dithiobis(2-nitrobenzoic acid).

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