Method for preparing aspergillus flavus uric acid oxidase by intracellular expression of yeast

Abstract

The present application relates to a method for preparing aspergillus flavus uric acid oxidase by intracellular expression of yeast, comprising the following steps: after inducing the intracellular expression of recombinant yeast to express aspergillus flavus uric acid oxidase, the cell is placed in a cell disruption buffer to disrupt the cell; wherein the cell disruption buffer contains an additive selected from one or a combination of dithiothreitol, tris (2-carboxyethyl) phosphine, cysteine and glutathione. The present application optimizes the homogenate buffer to inhibit the degradation of intracellular protein of yeast, ensures the integrity of the target protein, and also improves the cell disruption conditions such as reducing the homogenate pressure, thereby reducing the energy consumption and the requirements for equipment.

Description

Technical Field

[0001] This invention belongs to the field of protein expression and separation and purification, and specifically relates to a method for preparing aflatoxin urate oxidase by intracellular expression in yeast. Background Technology

[0002] Urate oxidase is an enzyme involved in purine metabolism in living organisms, catalyzing the reaction that converts uric acid into allantoin. Humans lost urate oxidase during evolution, rendering them unable to further break down uric acid. Furthermore, due to the poor solubility of uric acid, its excretion is difficult, leading to a predisposition to diseases caused by uric acid accumulation.

[0003] Tumor lysis syndrome (TLS) is a complication that occurs when the potassium, phosphorus, nucleic acid, and cytokines produced by the lysis of a large number of tumor cells due to chemotherapy exceed the body's self-regulating mechanisms. Because the human body lacks endogenous uricase, the uric acid produced by the release of nucleic acid metabolism rises sharply, easily exceeding the kidneys' excretion capacity. This leads to urate crystals obstructing the renal tubules, inducing acute kidney injury, and causing a series of subsequent symptoms. TLS is most common during chemotherapy for hematologic malignancies, but it can also occur during chemotherapy for other solid tumors. The development of TLS severely impacts patient prognosis and can potentially lead to life-threatening acute renal failure.

[0004] As early as the last century, researchers began treating TLS by injecting exogenous uricase. The one developed by Sanofi was launched in France in 1974. It was extracted from its naturally occurring fungus, Aspergillus flavus. It has a good and rapid effect in lowering blood uric acid, and can prevent kidney damage or restore normal kidney function. However, Aspergillus flavus is not a highly efficient expression system, and it contains a lot of toxins, making it difficult to guarantee the purity of the extracted protein. In 2001, Sanofi developed the first recombinant Aspergillus flavus uric acid oxidase, rasburicase, which was launched in Europe and in the United States the following year. Rasburicase is a product of recombinant expression in an intracellular soluble form using the cDNA of the Aspergillus flavus uric acid oxidase gene cloned in the Saccharomyces cerevisiae expression system, and has higher purity. Coiffier et al. categorized patients into high-risk, intermediate-risk, and low-risk groups for TLS. Rasburicase was considered a first-line drug for the prevention and treatment of TLS in high-risk individuals and intermediate-risk individuals for whom allopurinol prophylaxis had failed (COIFFIER B, ALTMAN A, PUI CH, et al. Guidelines for the management of pediatric and adult tumor lysis syndrome: an evidence-based review[J]. J Clin Oncol, 2008, 26(16): 2767-78.).

[0005] Sanofi uses Saccharomyces cerevisiae to express rasburicase. However, due to insufficient dissolved oxygen, Saccharomyces cerevisiae produces ethanol, making high-density fermentation difficult, resulting in unsatisfactory expression levels and high cost. Numerous imitation studies have been conducted domestically, primarily utilizing prokaryotic expression systems. However, prokaryotic systems suffer from the lack of post-translational modification. Pichia pastoris is a rapidly developing expression system in recent years, possessing a relatively complete post-translational modification system, providing a foundation for expressing specific proteins. However, this expression method also faces some limitations. For example, because yeast cell walls are thicker than prokaryotic cells, chemical, enzymatic, ultrasonic, and freeze-thaw lysis methods are not suitable for industrial production. Mechanical methods are required to break down cells and release intracellular proteins. Among these, bead milling has many operating parameters, is complex to optimize, and has an effective energy exchange rate of only about 1% (Bioseparation Engineering, 3rd Edition, by Sun Yan); while high-pressure homogenization requires cell disruption pressures of 1800-2000 bar, placing high demands on equipment. In addition, intracellular proteins are prone to degradation during homogenization and disruption; the release of intracellular impurities such as extraneous proteins, nucleic acids, and lipids also poses challenges to the separation and purification of the target protein. Summary of the Invention

[0006] This invention provides a method for preparing aflatoxin uricase using intracellular yeast expression, by optimizing the cell disruption buffer to inhibit the degradation of aflatoxin uricase during cell disruption.

[0007] Therefore, the present invention adopts the following technical solution:

[0008] This invention provides a method for preparing aflatoxin urate oxidase using intracellular expression in yeast, comprising, after inducing intracellular expression of aflatoxin urate oxidase in recombinant yeast, placing the cells in a cell lysis buffer to lyse the cells; wherein the cell lysis buffer contains an additive selected from one or more of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine, cysteine, and glutathione; preferably, the additive is dithiothreitol and / or tris(2-carboxyethyl)phosphine.

[0009] In some embodiments, the concentration of the additive is from 100 mM to 2 M, preferably from 100 mM to 1800 mM, 100 mM to 1600 mM, 100 mM to 1400 mM, 100 mM to 1200 mM, 100 mM to 1000 mM, 100 mM to 800 mM, 100 mM to 700 mM, 100 mM to 600 mM, 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 100 mM to 350 mM, 100 mM to 300 mM, 20 0mM to 2M, 200mM to 1800mM, 200mM to 1600mM, 200mM to 1400mM, 200mM to 1200mM, 200mM to 1000mM, 200mM to 800mM, 200mM to 700mM, 200mM to 600mM, 200mM to 500mM, 200mM to 450mM, 200mM to 400mM, 200mM to 350mM, 250mM to 350mM, 200mM to 300mM, or 250mM to 300mM. In some specific embodiments, the concentration of the additive is 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 900mM, 1000mM, 1100mM, 1200mM, 1300mM, 1400mM, 1500mM, 1600mM, 1700mM, 1800mM, 1900mM, or 2000mM.

[0010] In some embodiments, the additive of the present invention is tris(2-carboxyethyl)phosphine. Further, the concentration of said tris(2-carboxyethyl)phosphine is 100 mM to 2 M, preferably 100 mM to 1800 mM, 100 mM to 1600 mM, 100 mM to 1400 mM, 100 mM to 1200 mM, 100 mM to 1000 mM, 100 mM to 800 mM, 100 mM to 700 mM, 100 mM to 600 mM, 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 100 mM to 350 mM, 100 mM to 300 mM, 2 M, etc. 00mM to 2M, 200mM to 1800mM, 200mM to 1600mM, 200mM to 1400mM, 200mM to 1200mM, 200mM to 1000mM, 200mM to 800mM, 200mM to 700mM, 200mM to 600mM, 200mM to 500mM, 200mM to 450mM, 200mM to 400mM, 200mM to 350mM, 250mM to 350mM, 200mM to 300mM, or 250mM to 300mM. In some specific embodiments, the concentration of the tris(2-carboxyethyl)phosphine is 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 900mM, 1000mM, 1100mM, 1200mM, 1300mM, 1400mM, 1500mM, 1600mM, 1700mM, 1800mM, 1900mM, or 2000mM.

[0011] In some embodiments, the additive is dithiothreitol. Further, the concentration of the dithiothreitol is from 100 mM to 2 M, preferably from 100 mM to 1800 mM, 100 mM to 1600 mM, 100 mM to 1400 mM, 100 mM to 1200 mM, 100 mM to 1000 mM, 100 mM to 800 mM, 100 mM to 700 mM, 100 mM to 600 mM, 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 100 mM to 350 mM, 100 mM to 300 mM, or 20 mM. 0mM to 2M, 200mM to 1800mM, 200mM to 1600mM, 200mM to 1400mM, 200mM to 1200mM, 200mM to 1000mM, 200mM to 800mM, 200mM to 700mM, 200mM to 600mM, 200mM to 500mM, 200mM to 450mM, 200mM to 400mM, 200mM to 350mM, 250mM to 350mM, 200mM to 300mM, or 250mM to 300mM. In some specific embodiments, the concentration of the dithiothreitol is 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 900mM, 1000mM, 1100mM, 1200mM, 1300mM, 1400mM, 1500mM, 1600mM, 1700mM, 1800mM, 1900mM, or 2000mM.

[0012] In some embodiments, the additive is dithiothreitol and tris(2-carboxyethyl)phosphine. Further, the concentration of the dithiothreitol or tris(2-carboxyethyl)phosphine is from 100 mM to 2 M, preferably from 100 mM to 1800 mM, 100 mM to 1600 mM, 100 mM to 1400 mM, 100 mM to 1200 mM, 100 mM to 1000 mM, 100 mM to 800 mM, 100 mM to 700 mM, 100 mM to 600 mM, 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 100 mM to 350 mM, or 100 mM to 300 mM. mM, 200mM to 2M, 200mM to 1800mM, 200mM to 1600mM, 200mM to 1400mM, 200mM to 1200mM, 200mM to 1000mM, 200mM to 800mM, 200mM to 700mM, 200mM to 600mM, 200mM to 500mM, 200mM to 450mM, 200mM to 400mM, 200mM to 350mM, 250mM to 350mM, 200mM to 300mM, or 250mM to 300mM. In some specific embodiments, the concentration of the dithiothreitol or tris(2-carboxyethyl)phosphine is 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 900mM, 1000mM, 1100mM, 1200mM, 1300mM, 1400mM, 1500mM, 1600mM, 1700mM, 1800mM, 1900mM, or 2000mM.

[0013] In some embodiments, the buffering component of the cell lysis buffer of the present invention is tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), phosphate, borate-borax, or sodium barbital. In some embodiments, the concentration of the buffering component of the cell lysis buffer of the present invention is from 20 mM to 100 mM, preferably from 20 mM to 90 mM, 20 mM to 80 mM, 20 mM to 70 mM, 20 mM to 60 mM, 30 mM to 60 mM, 40 mM to 60 mM, 40 mM to 50 mM, or 50 mM to 60 mM. In some embodiments, the concentration of the buffering component of the cell lysis buffer of the present invention is 20 mM, 30 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the pH of the cell lysis buffer of the present invention is 8.0-8.5, 8.0-8.4, 8.0-8.3, 8.0-8.2, 8.0-8.1, or 8.0. In some embodiments, the buffering component of the cell lysis buffer of the present invention is 20 mM to 100 mM Tris-HCl, and the pH of the cell lysis buffer is 8.0-8.5; in one specific embodiment, the buffering component of the cell lysis buffer of the present invention is 50 mM Tris-HCl, and the pH of the cell lysis buffer is 8.0.

[0014] In some specific embodiments, after inducing intracellular expression of aflatoxin urate oxidase in recombinant yeast, the present invention places the bacterial cells in the cell lysis buffer to lyse the cells, and the resulting aflatoxin urate oxidase has a complete molecular weight.

[0015] In some embodiments, the present invention uses one or a combination of several of the following methods to disrupt cells: chemical method, osmotic pressure shock method, enzymatic method, ultrasonic method, freeze-thaw method, bead milling method, high-pressure homogenization method, and spray impact method; preferably, in some embodiments, the present invention uses high-pressure homogenization method to disrupt cells. In some embodiments, the present invention uses high-pressure homogenization method to homogenize cells one to five times. Preferably, in some embodiments, the present invention uses high-pressure homogenization method to homogenize cells one, two, three, four, or five times. In some embodiments, the homogenization pressure of the high-pressure homogenization method is 300 bar to 1800 bar, preferably 400 bar to 1800 bar, 450 bar to 1800 bar, 500 bar to 1800 bar, 600 bar to 1800 bar, 500 bar to 1600 bar, 500 bar to 1400 bar, 500 bar to 1200 bar, 500 bar to 1000 bar, 600 bar to 1000 bar, 500 bar to 800 bar, or 600 bar to 800 bar. In some embodiments, the homogenization pressure of the high-pressure homogenization method is 450 bar, 500 bar, 600 bar, 700 bar, 800 bar, 900 bar, 1000 bar, 1100 bar, 1200 bar, 1300 bar, 1400 bar, 1500 bar, 1600 bar, 1700 bar, or 1800 bar.

[0016] In some embodiments, the induced intracellular expression of aflatoxin urate oxidase in recombinant yeast according to the present invention includes the following steps: (a) cloning the gene encoding aflatoxin urate oxidase with an amino acid sequence as shown in SEQ ID NO:1 into an expression vector to construct a recombinant expression vector; (b) transforming the recombinant expression vector into yeast and screening to obtain recombinant yeast expressing aflatoxin urate oxidase; and (c) fermenting the recombinant yeast to induce the expression of aflatoxin urate oxidase.

[0017] In some embodiments, the yeast of the present invention is Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, or Candida albicans, preferably Pichia pastoris. In some embodiments, the Pichia pastoris of the present invention is GS115, KM71, KM71H, SMD1168, SMD1168H, or X33, preferably GS115.

[0018] In some embodiments, the nucleotide sequence of the gene encoding the aflatoxin urate oxidase described in this invention is shown in SEQ ID NO:2.

[0019] In some embodiments, the expression vector described in this invention is pPIC9K.

[0020] The construction of recombinant expression vectors and yeast transformation are common techniques in this field and can be carried out using conventional genetic engineering operations.

[0021] In some embodiments, the fermentation culture of recombinant yeast expressing aflatoxin urate oxidase can be carried out using conventional techniques in the art, such as seed activation using YPD solid medium, primary seed culture using YPD liquid medium, secondary seed culture using YPD liquid medium, and high-density fermentation culture in a fermenter using BSM medium.

[0022] In some embodiments, the method for preparing aflatoxin urate oxidase by intracellular expression of yeast provided by the present invention further includes isolating and purifying aflatoxin urate oxidase from the system after cell disruption.

[0023] In some embodiments, the method for isolating and purifying aflatoxin urate oxidase from the system after cell disruption provided by the present invention includes pretreatment of the homogenate obtained after cell disruption followed by anion exchange chromatography.

[0024] In some embodiments, the pretreatment of the present invention includes precipitation of the obtained homogenate. In some embodiments, the precipitation method is selected from one or a combination of two of polyethyleneimine precipitation and ammonium sulfate precipitation. In some embodiments, the precipitation method is a combination of polyethyleneimine precipitation and ammonium sulfate precipitation, for example, polyethyleneimine precipitation followed by ammonium sulfate precipitation; aflatoxin urate oxidase is present in the precipitate after ammonium sulfate precipitation. In some specific embodiments, in the polyethyleneimine precipitation of the present invention, the final concentration of polyethyleneimine in the system is 0.1% to 1.0% (w / v), preferably 0.1% to 0.8% (w / v), 0.1% to 0.6% (w / v), 0.1% to 0.4% (w / v), 0.1% to 0.3% (w / v), 0.1% to 0.2% (w / v), 0.2% to 0.4% (w / v), or 0.2% to 0.3% (w / v), more preferably 0.2% (w / v). In some specific embodiments, in the ammonium sulfate precipitation method of the present invention, the saturation of ammonium sulfate is 30% to 90% (w / v), preferably 40% to 90% (w / v), 50% to 90% (w / v), 50% to 80% (w / v), 50% to 70% (w / v), or 50% to 60% (w / v), more preferably 50% (w / v).

[0025] In some embodiments, after precipitation (e.g., after ammonium sulfate precipitation), the precipitate containing aflatoxin uricase is reconstituted with a reconstitution buffer to obtain a precipitate reconstitution solution, which is then replaced by ultrafiltration into the reconstitution buffer to obtain a replacement solution. In some embodiments, the buffering component of the reconstitution buffer of the present invention is phosphate, 2-(N-morpholino)ethanesulfonic acid, or Tris-HCl, preferably phosphate. In some embodiments, the phosphate concentration of the present invention is 5 mM to 50 mM, preferably 5 mM to 40 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 20 mM, 5 mM to 10 mM, or 10 mM. In some embodiments, the pH of the reconstitution buffer of the present invention is 7.0 to 9.7, preferably 7.0 to 9.0, 7.0 to 8.5, 7.5 to 8.2, or 8.2. In some embodiments, the ultrafiltration replacement of the present invention can be performed by a tubular membrane module, a flat-sheet membrane module, a spiral wound membrane module, or a hollow fiber tubular membrane module, preferably by a hollow fiber tubular membrane module. In some embodiments, the hollow fiber tubular membrane module has a molecular weight cutoff of 3 kDa to 100 kDa, preferably 3 kDa to 90 kDa, 3 kDa to 80 kDa, 3 kDa to 70 kDa, 3 kDa to 60 kDa, 3 kDa to 50 kDa, 5 kDa to 50 kDa, 10 kDa to 50 kDa, 20 kDa to 50 kDa, 30 kDa to 50 kDa, or 40 kDa to 50 kDa, more preferably 50 kDa.

[0026] In some embodiments, the chromatographic column packing material of the anion exchange chromatography of the present invention has a matrix of cellulose, dextran, or agarose, and the ion exchange group is diethylaminoethyl or quaternary ammonium. In some embodiments, the chromatographic column packing material of the present invention has a matrix of agarose, and the ion exchange group is diethylaminoethyl; specifically, for example, the chromatographic column packing material is DEAE-Sepharose FF.

[0027] In some embodiments, the chromatography column is pre-equilibrated before anion exchange chromatography. In some embodiments, the pH of the solution used for pre-equilibration is 8.0-9.7, preferably 8.2-8.7, and more preferably 8.2-8.3. In some embodiments, the buffer component of the solution used for pre-equilibration is phosphate or Tris-HCl, preferably Tris-HCl. In some embodiments, the concentration of the buffer component of the solution used for pre-equilibration is 5 mM to 100 mM, preferably 10 mM to 90 mM, 10 mM to 80 mM, 10 mM to 70 mM, 10 mM to 60 mM, 10 mM to 50 mM, 10 mM to 40 mM, 10 mM to 30 mM, or 10 mM to 20 mM, more preferably 20 mM.

[0028] The method for preparing aflatoxin uricase using intracellular yeast expression provided by this invention optimizes the cell disruption buffer, inhibits the degradation of aflatoxin uricase during cell disruption, and ensures the integrity of its sequence.

[0029] The method for preparing Aspergillus urate oxidase by intracellular expression of yeast provided by this invention optimizes the cell disruption buffer and unexpectedly found that it can improve cell disruption conditions, such as reducing homogenization pressure, while ensuring the release effect of Aspergillus urate oxidase, thereby reducing production energy consumption and reducing equipment requirements.

[0030] This invention utilizes intracellular expression of yeast to prepare aflatoxin uricase, ensuring the sequence consistency of the prepared aflatoxin uricase with the natural sequence, including consistency in post-translational modifications.

[0031] The method for preparing Aspergillus uricase using intracellular yeast expression provided by this invention has a relatively simple purification method, effectively removes impurities and proteins, and yields Aspergillus uricase with high purity and good enzyme activity. The Aspergillus uricase prepared using this method shows good consistency with Sanofi's commercial recombinant Aspergillus uricase. Attached Figure Description

[0032] Figure 1 This is a mass spectrum of Aspergillus urate oxidase obtained after homogenization and cell disruption using initial homogenization buffer and subsequent purification.

[0033] Figure 2 This is a mass spectrum of Aspergillus uricase oxidase obtained by homogenization and purification after cell disruption using homogenization buffer with 100 mM DTT as an additive.

[0034] Figure 3 This is a mass spectrum of Aspergillus uricase oxidase obtained after homogenization and cell disruption using homogenization buffer with 300 mM DTT as an additive and subsequent purification.

[0035] Figure 4 SDS-PAGE images of Sanofi commercial recombinant aspergillus urate oxidase and self-made recombinant aspergillus urate oxidase: Lane M: Marker; Lane 1: Sanofi commercial recombinant aspergillus urate oxidase; Lanes 2-7: Self-made recombinant aspergillus urate oxidase.

[0036] Figure 5 The RP-HPLC chromatograms of Sanofi's commercial recombinant aflatoxin uricase and its self-made recombinant aflatoxin uricase are shown. Detailed Implementation

[0037] Specific embodiments of the present invention

[0038] The present invention provides the following specific implementation schemes.

[0039] Implementation plan [1]: A method for preparing aflatoxin urate oxidase by intracellular expression of yeast, characterized in that it includes: after inducing intracellular expression of aflatoxin urate oxidase in recombinant yeast, placing the cells in a cell lysis buffer to break the cells; wherein the cell lysis buffer contains an additive, the additive being selected from one or a combination of several of dithiothreitol, tris(2-carboxyethyl)phosphine, cysteine, and glutathione.

[0040] Implementation Scheme [2]: The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to Implementation Scheme [1] is characterized in that the additive is dithiothreitol and / or tris(2-carboxyethyl)phosphine.

[0041] Implementation Scheme [3]: The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to Implementation Scheme [1] is characterized in that the concentration of the additive is 100mM to 2M, preferably 200mM to 2M, and more preferably 300mM.

[0042] Implementation Scheme [4]: ​​The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to Implementation Scheme [2] is characterized in that the concentration of dithiothreitol or tris(2-carboxyethyl)phosphine is 100mM to 2M, preferably 200mM to 2M, and more preferably 300mM.

[0043] Implementation Scheme [5]: The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to any one of the implementation schemes [1]-[4] is characterized in that the buffering components of the cell disruption buffer are tris(hydroxymethyl)aminomethane-hydrochloric acid, phosphate, boric acid-borax or sodium barbital; and the pH of the cell disruption buffer is 8.0-8.5.

[0044] Implementation Scheme [6]: The method for preparing aflatoxin uric acid oxidase by intracellular expression of yeast according to any one of the implementation schemes [1]-[5] is characterized in that the cell disruption is performed by one or a combination of chemical method, osmotic pressure shock method, enzymatic method, ultrasonic method, freeze-thaw method, bead milling method, high pressure homogenization method, and spray impact method; preferably, the cell disruption is performed by high pressure homogenization method.

[0045] Implementation scheme [7]: According to the method for preparing aflatoxin urate oxidase by intracellular expression of yeast as described in implementation scheme [6], the homogenization pressure of the high-pressure homogenization method is 300 bar to 1800 bar, preferably 600 bar to 1800 bar.

[0046] Implementation Scheme [8]: The method for preparing aflatoxin uricase by intracellular expression of yeast according to any one of Implementation Schemes [1]-[7] is characterized in that inducing intracellular expression of aflatoxin uricase in recombinant yeast includes the following steps:

[0047] a) The gene encoding the amino acid sequence of Aspergillus uricase as shown in SEQ ID NO:1 was cloned into an expression vector to construct a recombinant expression vector;

[0048] b) Transform the recombinant expression vector into yeast and screen for recombinant yeast expressing aflatoxin uricase; and

[0049] c) Fermentation culture of recombinant yeast to induce expression of aflatoxin urate oxidase.

[0050] Implementation scheme [9]: The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to implementation scheme [8] is characterized in that the expression vector is pPIC9K.

[0051] Implementation Scheme

[10] : The method for preparing aflatoxin uric acid oxidase by intracellular expression of yeast according to any one of the implementation schemes [1]-[9] is characterized in that the yeast is Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha or Candida albicans, preferably Pichia pastoris.

[0052] Implementation Scheme

[11] : The method for preparing aflatoxin uric acid oxidase by intracellular expression of yeast according to Implementation Scheme

[10] is characterized in that the Pichia pastoris is GS115, KM71, KM71H, SMD1168, SMD1168H or X33, preferably GS115.

[0053] Implementation scheme

[12] : The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to any one of the implementation schemes [1]-

[11] is characterized in that it further includes isolating and purifying aflatoxin urate oxidase from the system after cell lysis.

[0054] definition

[0055] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art.

[0056] The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to an expression system (e.g., a host cell or an in vitro expression system). One type of vector is a "plasmid," which refers to a circular double-stranded DNA (dsDNA) molecule into which other DNA fragments can be inserted. Another type of vector is a viral vector, in which other DNA fragments can be inserted into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) can be integrated into the genome of the host cell after introduction, thereby replicating along with the host genome. According to some embodiments of the invention, the vector is a vector for gene cloning. According to some embodiments of the invention, the vector is a vector for gene expression.

[0057] The term "expression vector" refers to a vector that can be used to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector in a biological or regenerated biological system. According to some embodiments of the invention, the expression vector is an *Escherichia coli* expression vector. According to some embodiments of the invention, the expression vector is the *Pichia pastoris* expression vector pPIC9K.

[0058] The term "transformation" refers to the process of introducing a foreign nucleic acid fragment into a host cell, thereby altering its genetic traits. A host cell that has undergone transformation, containing the foreign nucleic acid fragment and exhibiting altered genetic traits, is called a "transformant." Vectors can be transferred into host cells using transformation methods known in the art. According to some embodiments of the present invention, the transformation methods include methods known in the art such as electroconversion and chemical-mediated transformation. According to some embodiments of the present invention, the transformation method is electroconversion.

[0059] The term "cell disruption buffer" as used in this article refers to a buffer solution used to suspend host cells during cell disruption. It generally includes buffering components to maintain pH stability during the cell disruption process. Additionally, some non-standard components may be added as "additives" to the cell disruption buffer to achieve different cell disruption effects. When high-pressure homogenization is used to disrupt cells, the "cell disruption buffer" can also be called a "homogenization buffer."

[0060] The term "homogeneous material" as used in this article refers to the process of lysing or mechanically lysing host cells to release intracellular substances from the host cells. The system containing lysed host cells and intracellular substances obtained after homogenizing and disrupting cells is called a "homogeneous solution".

[0061] The term "deconvolution" refers to using mathematical algorithms to re-analyze unseparated chromatographic components, restoring their most accurate mass spectrometric information. The total ion current chromatogram (TIC) is actually fitted by software based on multiple data points acquired by mass spectrometry. Each data point corresponds to a mass spectrum. When two or more chromatographic peaks co-elute without separation, the acquired mass spectrometry data points result in a mixed mass spectrum containing fragmented information from multiple components. Directly using this for qualitative analysis can lead to reduced substance similarity and component loss.

[0062] An enzyme activity unit (U) in this article is defined as the amount of enzyme required to consume 1 μmol of uric acid per minute in a 0.1 M borate buffer (pH 8.9) system at a temperature of 30°C.

[0063] The term "specific activity" refers to the number of enzyme activity units contained per milligram (mg) of protein.

[0064] The present invention will be further described below with reference to specific embodiments. However, these embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the present invention is not limited to any specific preferred embodiments described herein. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the technical features of the present invention still fall within the protection scope of the present invention. Unless otherwise specified, the reagents used in the following embodiments are all commercially available products, and the solutions can be prepared using conventional techniques in the art.

[0065] Unless otherwise specified, the operation of this invention will be performed using conventional techniques within the scope of the art, such as biochemistry and protein purification, or in accordance with the product instructions, which are fully explained in the literature. In the following examples, efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, times, etc.), but some experimental errors and biases should be taken into account.

[0066] Example 1: Determination of the enzyme activity of recombinant Aspergillus flavus uricase oxidase by ultraviolet spectrophotometry

[0067] Uric acid, the substrate of Aspergillus flavus uricase, has maximum absorption at 292 nm. Therefore, the decrease in absorbance at the detection wavelength of 292 nm in the reaction system can represent enzyme activity. A 0.1 M borate buffer (pH 8.9) was prepared to dissolve uric acid to a concentration of 20 μg / ml as the working substrate solution. The reaction system is shown in the table below. After adding the reaction system to a 96-well plate, it was placed in a microplate reader at 30℃. The absorbance was measured at the detection wavelength of 292 nm every 30 seconds. Enzyme activity, i.e., the initial rate of enzyme reaction, is reflected by the rate of decrease in absorbance at the detection wavelength of 292 nm measured at time points 2-5. Simultaneously, the absorbance values ​​of borate buffer containing different molar concentrations of uric acid at the detection wavelength of 292 nm were measured, and a molar concentration-OD plot was plotted. 292 nm The enzyme activity was calculated by using the standard curve and the rate of decrease of the absorbance value measured above.

[0068] Element volume substrate working fluid 150μl Aspergillus urate oxidase solution 50μl

[0069] Example 2: Construction of recombinant Pichia pastoris expressing aflatoxin uricase oxidase

[0070] The steps for strain construction are as follows:

[0071] (1) Based on the codon preference of Pichia pastoris, the amino acid sequence of Aspergillus flavus urate oxidase as shown in SEQ ID NO:1 was converted into a DNA sequence as shown in SEQ ID NO:2. BamHⅠ-EcoRⅠ restriction endonuclease sites were added to the end, and the gene was synthesized and amplified by PCR.

[0072] (2) The amplified gene and pPIC9K (Invitrogen) were digested with BamHI-EcoRI, and the digestion mixture was separated and recovered by 0.8% agarose gel electrophoresis and ligated with T4 ligase.

[0073] (3) The enzyme-linked product was electroporated into E. coli DH5α competent cells. After recovery culture, the bacterial culture was taken to extract plasmids. The extracted plasmids were linearized by enzyme digestion at the SalⅠ site. The enzyme digestion mixture was separated and recovered by 0.8% agarose gel electrophoresis.

[0074] (4) The recovered linearized plasmid was electroporated into the host cell Pichia pastoris GS115 strain (Invitrogen) and then revived and cultured.

[0075] (5) The GS115 cell mixture after resuscitation was spread on histidine auxotrophic plates (MD solid medium) and transformants were screened; the MD solid medium formula was: amino-free yeast nitrogen source 13.4 g / L, glucose 20 g / L, biotin 4 mg / L, agarose 25 g / L.

[0076] (6) Transformants were picked from histidine auxotrophic plates and fermented in shake flasks to screen recombinant Pichia pastoris transformants that efficiently expressed Aspergillus urate oxidase as starting strains.

[0077] Example 3: Fermentation culture of the starting strain and induction expression of uricase from Aspergillus flavus

[0078] The starting strain was fermented at high density in a 5L fermenter. The culture medium and trace element (PTM1) formulations are as follows:

[0079] YPD solid medium: tryptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar powder 20 g / L;

[0080] YPD liquid culture medium: tryptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L;

[0081] BSM medium: phosphate 26.7 ml / L, calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate heptahydrate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, PTM1 4.35 ml / L;

[0082] PTM1: Copper sulfate pentahydrate 6g / L, sodium iodide 0.08g / L, manganese sulfate monohydrate 3g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc chloride 20g / L, ferrous sulfate heptahydrate 65g / L, biotin 0.2g / L, sulfuric acid 5ml / L;

[0083] Glycerol-supplemented medium: 75% (w / v) glycerol, PTM1 12 ml / L;

[0084] Methanol-fed medium: a methanol solution containing 12 ml / L of PTM1.

[0085] Under aseptic conditions, bacterial culture was extracted from a glycerol tube containing the starting strain using an inoculation loop and streaked onto YPD solid medium for seed activation. After incubation at 30°C for 48 hours, single colonies were aseptically picked and inoculated into a shake flask containing 30 ml of YPD liquid medium for primary seed culture. After incubation at 220 rpm and 30°C for 24 hours, a 10% (v / v) inoculation was carried out into a shake flask containing YPD liquid medium for secondary seed culture. After incubation at 220 rpm and 30°C for 24 hours, a 10% (v / v) inoculation was carried out into a 5L fermenter containing 2.5 L of BSM medium for high-density fermentation. The fermentation temperature was controlled at 30°C, dissolved oxygen at 40%, and pH at 5.0. After 12-14 hours of culture, glycerol-feeding medium was added to initiate glycerol feeding at a rate of 22.5 ml / (h·L fermentation broth) over approximately 4 hours. After glycerol feeding, the culture medium was starved of carbon source for approximately 1 hour before methanol induction was initiated. A stepped increase in the methanol feeding rate was adopted, specifically: for the first hour of induction, the feed rate was manually added dropwise at less than 3.6 ml / (h·L fermentation broth); from hour 1 to hour 8, the feed rate was 5.0 ml / (h·L fermentation broth); from hour 8 to hour 22, the feed rate was 10.0 ml / (h·L fermentation broth); and from hour 22 to hour 48, the feed rate was 12.0 ml / (h·L fermentation broth). After approximately 48 hours of induction, the culture was transferred to a container, and the cells were collected by centrifugation.

[0086] Example 4: Isolation and purification of uricase from Aspergillus flavus

[0087] (1) High-pressure homogenization method to disrupt recombinant Pichia pastoris cells

[0088] Recombinant Pichia pastoris cells were homogenized using a JN-02C (Juning Bio) cell disruptor. The cells collected by centrifugation in Example 3 were resuspended in wash buffer (5 mM Tris-HCl, pH 8.0) (5 ml wash buffer / g cells). After resuspending, the supernatant was removed by centrifugation, and the precipitate was resuspended in homogenization buffer (10 ml homogenization buffer / g cells). The recombinant Pichia pastoris cells were homogenized twice at a homogenization pressure of 1800 bar. The homogenate was collected, and the supernatant was collected by centrifugation.

[0089] (2) Pretreatment: Polyethyleneimine precipitation and ammonium sulfate precipitation

[0090] Under stirring, a 10% (w / v) polyethyleneimine (PEI) solution, pre-adjusted to pH 8.0 with phosphoric acid, was slowly added dropwise to the supernatant of the homogenate until the final concentration of PEI in the homogenate was 0.2% (w / v). After precipitation equilibration for half an hour, the supernatant was collected by centrifugation. Then, under stirring, ammonium sulfate powder was added to the supernatant until the ammonium sulfate saturation was 50% (w / v). After precipitation equilibration for half an hour, the precipitate was collected by centrifugation. The precipitate was reconstituted with reconstitution buffer (10 mM Na₂HPO₄, pH 8.2) to obtain a precipitate reconstitution solution.

[0091] (3) Anion exchange chromatography

[0092] A chromatography column (GE Healthcare) packed with DEAE-Sepharose FF packing material was pre-equilibrated with 20 mM Tris-HCl (pH 8.2) for at least 10 column volumes. The precipitate was completely replaced with reconstitution buffer (10 mM Na₂HPO₄, pH 8.2) using a 50 kDa hollow fiber ultrafiltration membrane. The solution was then filtered through a 0.22 μm aqueous filter and loaded onto the sample. The flow-through was collected. The collected flow-through was then desalted by replacing the precipitate with 10 mM Na₂HPO₄ (pH 8.0) using a 50 kDa hollow fiber ultrafiltration membrane to obtain a batch of purified Aspergillus flavus urate oxidase solution.

[0093] Example 5: Mass spectrometry identification of uricase from Aspergillus flavus

[0094] The purified uric acid oxidase solution from *Aspergillus flavus* was analyzed by LC-MS. The mass spectrometer used was a Waters Xevo G2-XS QTof high-resolution mass spectrometer. The ultra-high performance liquid chromatograph (UPLC) system was a Waters ACQUITY UPLC system; the chromatographic column was a YMCTriartbio C4 column; the aqueous phase was an aqueous solution containing 0.1% (v / v) formic acid, and the organic phase was an acetonitrile solution containing 0.1% (v / v) formic acid. Specific detection parameters are shown in the table below.

[0095] Sample injection 5μl Column temperature 50℃ Flow rate 0.4 ml / min time 10min-15min gradient 5%-95% organic phase

[0096] The raw data files obtained from mass spectrometry analysis can be deconvolved using Unidec software.

[0097] Example 6: Homogenization, separation, purification, and mass spectrometry detection using the initial homogenization buffer formulation.

[0098] First, an initial homogenization buffer (50 mM Tris-HCl, pH 8.0) was prepared. Aspergillus urate oxidase was isolated and purified according to the method in Example 4. The purified aspergillus urate oxidase with a purity of 95%-96% was analyzed by mass spectrometry according to the method in Example 5.

[0099] Mass spectrometry analysis results as follows Figure 1 As shown. The deconvolution calculation of the complete molecular weight is 34038 Da, which is exactly one leucine residue (113 Da) different from the molecular weight of the aspergillus uricase protein (34151 Da) shown in SEQ ID NO:1. The first amino acid residue at the C-terminus of the recombinant aspergillus uricase protein is a leucine residue. Therefore, it can be determined that homogenization using the homogenization buffer prepared according to the initial formula cannot yield the complete aspergillus uricase protein.

[0100] Example 7: Homogenization, separation, purification, and mass spectrometry detection using homogenization buffers with different additives.

[0101] The homogenization buffer, with the components shown in the table below, was prepared and the pH adjusted with hydrochloric acid. Disodium ethylenediaminetetraacetate (EDTA-2Na) and the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) are additives mentioned in the *Protein Purification Guide* for inhibiting protein degradation during homogenization. The concentrations used were in accordance with or higher than those recommended in the guide. Since recombinant *Aspergillus flavus* uricase does not contain disulfide bonds, dithiothreitol (DTT) was also considered as an additive in the homogenization buffer to protect intracellular products. *Aspergillus flavus* uricase was isolated and purified according to the method in Example 4. The purified *Aspergillus flavus* uricase with a purity of 95%-96% was analyzed by mass spectrometry according to the method in Example 5.

[0102]

[0103] Mass spectrometry deconvolution results showed that using EDTA-2Na or AEBSF as homogenate buffer additives yielded aflatoxin-degrading uric acid oxidase with a molecular weight of 34038 Da. However, when DTT was used as an additive, the mass spectrometry results were as follows: Figure 2 As shown, deconvolution analysis of the mass spectrometry data revealed that the degradation product with a molecular weight of 34038 Da accounted for approximately 35%, while the remainder consisted of intact Aspergillus flavus uricase with a molecular weight of 34151 Da. This demonstrates that DTT can partially inhibit degradation when used as a homogenization buffer additive.

[0104] Example 8: Homogenization, separation, purification, and mass spectrometry detection using homogenization buffers with different reducing agents.

[0105] Prepare a homogenization buffer solution with the components shown in the table below, adjust the pH with hydrochloric acid, and add additives such as the reducing agent tris(2-carboxyethyl)phosphine (TCEP), cysteine ​​(Cys), or DTT. The aflatoxin uricase was isolated and purified according to the method in Example 4. The purified aflatoxin uricase solution with a purity of 95%-96% was analyzed by mass spectrometry according to the method in Example 5.

[0106]

[0107] Mass spectrometry deconvolution results showed that using TCEP as a homogenization buffer additive yielded similar results to using DTT. However, using Cys as a homogenization buffer additive resulted in an impurity with a molecular weight of 34270 Da. Using reducing agents such as TCEP or DTT as homogenization buffer additives effectively inhibited degradation, yielding recombinant aflatoxin uricase with its complete molecular weight.

[0108] Example 9: Homogenization, separation, purification, and mass spectrometry detection using homogenization buffers with different concentrations of DTT.

[0109] Prepare a homogenization buffer solution with the components shown in the table below, and adjust the pH with hydrochloric acid. Isolate and purify Aspergillus urate oxidase according to the method in Example 4. The obtained purified Aspergillus urate oxidase solution with a purity of 95%-96% was analyzed by mass spectrometry according to the method in Example 5.

[0110]

[0111] Mass spectrometry deconvolution results showed that when DTT was used as a homogenization buffer additive, deconvolution analysis detected few degradation products at a concentration of 200 mM, and no degradation products were detected at a concentration of 300 mM. The mass spectrometry results are as follows. Figure 3 As shown.

[0112] Example 10: Optimization of homogenization conditions for disrupted recombinant Pichia pastoris cells

[0113] First, the homogenization pressure under the condition of adding DTT was optimized. Referring to the method of "(1) High pressure homogenization to break recombinant Pichia pastoris cells" in Example 4, under the condition of 50mM Tris-HCl, 300mM DTT, and pH 8.0 as homogenization buffer components, homogenization was performed twice at homogenization pressures of 300 bar, 450 bar, 600 bar, 1200 bar, or 1800 bar. The supernatant of the homogenate was directly taken for enzyme activity determination according to the method of Example 1. The homogenate was diluted 2000 times before the determination. The results are shown in the table below. The relative enzyme activity is defined as the ratio of the enzyme activity of the homogenate obtained under other homogenization pressures to the enzyme activity of the homogenate obtained under a homogenization pressure of 1800 bar.

[0114] Homogenization pressure Relative enzyme activity 1800 bar 100％ 1200 bar 96％ 600 bar 83％ 450 bar 58％ 300 bar 46％

[0115] Enzyme activity assays showed that the release of intracellular Aspergillus flavus uricase was similar at homogenization pressures of 1800 bar, 1200 bar, and 600 bar, but the release was poorer when homogenizing at lower pressures. This means that reducing the homogenization pressure to 600 bar can also recover more Aspergillus flavus uricase, thus reducing the need for equipment.

[0116] Next, the effect of adding DTT on the release of intracellular Aspergillus flavus uricase was compared. Referring to the method of "(1) high-pressure homogenization to disrupt recombinant Pichia pastoris cells" in Example 4, 50 mM Tris-HCl, pH 8.0, or 50 mM Tris-HCl, 300 mM DTT, pH 8.0 were used as homogenization buffer components. Homogenization was performed twice at a pressure of 600 bar. The supernatant of the homogenate was directly taken for enzyme activity determination according to the method in Example 1. The homogenate was diluted 2000 times before determination. The results are shown in the table below. The relative enzyme activity was defined as the ratio of the enzyme activity of the homogenate obtained under homogenization conditions of 50 mM Tris-HCl, pH 8.0 to the enzyme activity of the homogenate obtained under homogenization conditions of 50 mM Tris-HCl, 300 mM DTT, pH 8.0.

[0117] Homogenization buffer Relative enzyme activity 50mM Tris-HCl, pH 8.0 65％ 50mM Tris-HCl, 300mM DTT, pH 8.0 100％

[0118] Enzyme activity assays showed that adding 300 mM DTT as a homogenate buffer additive significantly improved the release of intracellular Aspergillus flavus uricase oxidase compared to the absence of additives.

[0119] Example 11: Determination of the purity of recombinant Aspergillus flavus uricase oxidase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0120] The homogenization buffer formulation was 50 mM Tris-HCl, 300 mM DTT, pH 8.0. The homogenization pressure was 600 bar, and the homogenization was performed twice. Other purification procedures followed those in Example 4 to obtain the self-prepared recombinant aflatoxin uricase. The purity of the purified recombinant aflatoxin uricase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 12.5% ​​gel was prepared according to the method in *Protein Purification Guidelines*, and reduction electrophoresis was performed. The detection results are as follows... Figure 4 As shown, the sample exhibits a single band on 12.5% ​​SDS-PAGE electrophoresis, indicating high purity. Furthermore, the band position is consistent with that of the Sanofi commercial recombinant aflatoxin uricase oxidase, demonstrating good consistency.

[0121] Example 12: Purity determination of recombinant Aspergillus flavus uricase by reversed-phase high-performance liquid chromatography

[0122] The homogenization buffer formulation was 50 mM Tris-HCl, 300 mM DTT, pH 8.0. The homogenization pressure was 600 bar, and the homogenization was performed twice. Other purification procedures followed those in Example 4 to obtain the self-made recombinant aflatoxin uric acid oxidase. Purity was determined using reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC system used was a Waters ACQUITY HPLC system; the column used was a YMC Triartbio C4 column; the aqueous phase (phase A) was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and the organic phase (phase B) was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. Specific detection parameters are shown in the table below.

[0123] Sample injection 5μg Column temperature 60℃ Flow rate 1ml / min time 33min

[0124] The gradients are shown in the table below.

[0125] Time (min) Phase A proportion (%) Phase B percentage (%) 0 80 20 6 80 20 26 40 60 27 80 20 33 80 20

[0126] Test results as follows Figure 5 As shown in the figure, it can be determined that the target peak is present on the RP-HPLC chromatogram of the self-made recombinant aflatoxin uric acid oxidase. It has good symmetry, no other impurity peaks, high purity, and the retention time is consistent with that of Sanofi's commercial recombinant aflatoxin uric acid oxidase Rasburicase, proving that the two have good consistency.

[0127] Example 13: Determination of the specific activity of recombinant aflatoxin uricase oxidase

[0128] The homogenization buffer formulation was 50 mM Tris-HCl, 300 mM DTT, pH 8.0. The homogenization pressure was 600 bar, and the homogenization was performed twice. Other purification procedures followed those in Example 4 to obtain the self-made recombinant aspergillus urate oxidase. The self-made recombinant aspergillus urate oxidase and the commercially available Sanofi recombinant aspergillus urate oxidase were prepared into 1 μg / ml solutions, and their enzyme activities were determined according to the method in Example 1. Based on the enzyme activity determination results, the specific activity calculation results are shown in the table below.

[0129] sample Specific activity (U / mg) Homemade recombinant aflatoxin uricase 11.6 Sanofi's recombinant aflatoxin urate oxidase 11.7

[0130] The results showed that the specific activity of the self-made recombinant aflatoxin urate oxidase was basically the same as that of Sanofi's commercial recombinant aflatoxin urate oxidase Rasburicase, indicating that it had good enzyme activity. sequence list <110> Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Shanghai Institute of Pharmaceutical Industry Shanghai Domir Biotechnology Co., Ltd. <120> Method for preparing aflatoxin uricase by intracellular expression in yeast <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 301 <212> PRT <213> Aspergillus flavus <400> 1 Ser Ala Val Lys Ala Ala Arg Tyr Gly Lys Asp Asn Val Arg Val Tyr 1 5 10 15 Lys Val His Lys Asp Glu Lys Thr Gly Val Gln Thr Val Tyr Glu Met 20 25 30 Thr Val Cys Val Leu Leu Glu Gly Glu Ile Glu Thr Ser Tyr Thr Lys 35 40 45 Ala Asp Asn Ser Val Ile Val Ala Thr Asp Ser Ile Lys Asn Thr Ile 50 55 60 Tyr Ile Thr Ala Lys Gln Asn Pro Val Thr Pro Pro Glu Leu Phe Gly 65 70 75 80 Ser Ile Leu Gly Thr His Phe Ile Glu Lys Tyr Asn His Ile His Ala 85 90 95 Ala His Val Asn Ile Val Cys His Arg Trp Thr Arg Met Asp Ile Asp 100 105 110 Gly Lys Pro His Pro His Ser Phe Ile Arg Asp Ser Glu Glu Lys Arg 115 120 125 Asn Val Gln Val Asp Val Val Glu Gly Lys Gly Ile Asp Ile Lys Ser 130 135 140 Ser Leu Ser Gly Leu Thr Val Leu Lys Ser Thr Asn Ser Gln Phe Trp 145 150 155 160 Gly Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp Arg 165 170 175 Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Asn Phe Ser 180 185 190 Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr Trp 195 200 205 Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp Asn Ser 210 215 220 Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu Gln Ile Leu Ala 225 230 235 240 Arg Gln Gln Leu Ile Glu Thr Val Glu Tyr Ser Leu Pro Asn Lys His 245 250 255 Tyr Phe Glu Ile Asp Leu Ser Trp His Lys Gly Leu Gln Asn Thr Gly 260 265 270 Lys Asn Ala Glu Val Phe Ala Pro Gln Ser Asp Pro Asn Gly Leu Ile 275 280 285 Lys Cys Thr Val Gly Arg Ser Ser Leu Lys Ser Lys Leu 290 295 300 <210> 2 <211> 903 <212> DNA <213> Artificial Sequence <400> 2 tcagctgtaa aggctgccag atacggtaag gacaacgtta gagtctacaa ggttcacaag 60 gacgaaaaga ctggtgtcca aactgtctac gagatgactg tttgtgtctt gttggagggt 120 gaaatcgaga cttcttacac taaggccgac aactccgtta tcgttgctac tgattccatc 180 aagaacacta tctacatcac tgccaagcaa aacccagtta ctccaccaga attgttcggt 240 tctatcttg gtactcactt catcgagaag tacaaccaca tccacgctgc ccacgttaac 300 atcgtctgtc acagatggac tagaatggat atcgacggta agccacaccc acactctttc 360 atcagagatt ccgaagagaa gagaaacgtc caggttgatg ttgtcgaagg taagggtatc 420 gacatcaagt cttccttgtc tggtttgact gtcttgaagt ctactaactc tcaattctgg 480 ggtttcttga gagacgagta cactactttg aaggaaactt gggacagaat cttgtctact 540 gatgtcgatg ctacttggca atggaagaac ttctctggtt tgcaagaggt cagatcccac 600 gttccaaagt tcgatgccac ttgggctact gctagagagg ttactttgaa gactttcgcc 660 gaagataact ccgcttctgt tcaagccact atgtacaaga tggccgaaca aatcttggcc 720 agacaacagt tgatcgagac tgttgaatac tccttgccaa acaagcacta cttcgagatc 780 gacttgtcgt ggcacaaggg tctccaaaac actggcaaga acgctgaagt cttcgctcca 840 caatctgatc caaacggtttt gatcaagtgt actgttggta gatcctcctt gaagtccaag 900 tg 903

Claims

1. A method for preparing aflatoxin uricase oxidase using intracellular expression in yeast, characterized in that, include: After inducing intracellular expression of aflatoxin urate oxidase in recombinant yeast, the cells were placed in a cell lysis buffer to rupture them. The cell lysis buffer contains an additive, namely dithiothreitol, at a concentration of 300 mM.

2. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 1, characterized in that, The buffering components of the cell lysis buffer are tris(hydroxymethyl)aminomethane-hydrochloric acid, phosphate, borate-borax, or sodium barbital; the pH of the cell lysis buffer is 8.0-8.

5.

3. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 1, characterized in that, Cell disruption is achieved using one or a combination of methods, including chemical methods, osmotic pressure shock methods, enzymatic methods, ultrasonic methods, freeze-thaw methods, bead milling methods, high-pressure homogenization methods, and spray impact methods.

4. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 3, characterized in that, Cell disruption was achieved using a high-pressure homogenization method.

5. The method for preparing aflatoxin urate oxidase using intracellular yeast expression according to claim 4, characterized in that, The homogenization pressure of the high-pressure homogenization method is 300 bar to 1800 bar.

6. The method for preparing aflatoxin urate oxidase using intracellular yeast expression according to claim 5, characterized in that, The homogenization pressure of the high-pressure homogenization method is 600 bar to 1800 bar.

7. The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to any one of claims 1-6, characterized in that, Inducing intracellular expression of aflatoxin uricase in recombinant yeast involves the following steps: a) The gene encoding the amino acid sequence of Aspergillus uricase as shown in SEQ ID NO:1 was cloned into an expression vector to construct a recombinant expression vector; b) Transform the recombinant expression vector into yeast and screen for recombinant yeast expressing aflatoxin uricase; and c) Fermentation culture of recombinant yeast to induce expression of aflatoxin urate oxidase.

8. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 7, characterized in that, The expression vector is pPIC9K.

9. The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to any one of claims 1-6, characterized in that, The yeast is Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, or Candida albicans.

10. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 9, characterized in that, The yeast in question is Pichia pastoris.

11. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 10, characterized in that, The Pichia pastoris is GS115, KM71, KM71H, SMD1168, SMD1168H or X33.

12. The method for preparing aflatoxin uricase using intracellular yeast expression according to claim 11, characterized in that, The Pichia pastoris is GS115.

13. The method for preparing aflatoxin urate oxidase by intracellular expression of yeast according to any one of claims 1-6, characterized in that, It also includes the isolation and purification of aflatoxin urate oxidase from the system after cell disruption.

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