Nucleic acid molecule for coding urate oxidase, related product and application of nucleic acid molecule

Abstract

The invention discloses a nucleic acid molecule for coding urate oxidase, a related product and an application of the nucleic acid molecule. The invention discloses a nucleic acid molecule for coding urate oxidase. The nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO. 2. According to the invention, a microbe-derived urate oxidase gene sequence for escherichia coli expression is obtained for the first time, and the gene sequence is easy to clone and express and good in repeatability. The invention further discloses a recombinant vector and a bioengineering bacterium for urate oxidase expression, and the obtained urate oxidase protein is high in activity and simple to prepare.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a nucleic acid molecule encoding urate oxidase, related products and their uses. Background Art

[0002] Purine compounds, with the basic molecular formula C5H4N4, are a class of compounds containing a nitrogen heterocyclic structure and are metabolites in the process of metabolism. The common ones in the human body include adenine, guanine, hypoxanthine and xanthine. At the same time, we also ingest purine substances through diet. In the human body, purines undergo a series of reactions and are finally oxidized to form the end product uric acid.

[0003] The synthesis of uric acid mainly occurs in the human body through the metabolic process of purines. The metabolism of uric acid mainly occurs in the kidneys. The kidneys excrete uric acid out of the body through glomerular filtration. Under normal circumstances, almost all of the uric acid filtered by the glomeruli is reabsorbed by the renal tubules and transported back to the liver for reuse.

[0004] Uricase (uricase, urate oxidase), also called urate oxidase, is an oxidase involved in the purine degradation pathway and can decompose uric acid into more soluble allantoin, CO2 and H2O2, making it easier for uric acid to be excreted from the body. During the evolution of humans and other primates, the uricase gene has gradually been lost. Therefore, humans cannot produce active uricase like other animals. Due to the lack of uricase, uric acid has become the end product of purine metabolism and is prone to accumulate in the body. When the fasting blood uric acid level is higher than 420 μmol / L in men and higher than 360 μmol / L in women on two non-consecutive days, it is called hyperuricemia. Excess uric acid will deposit in joints, soft tissues, cartilage and kidneys in the form of crystals, causing diseases such as gout, arthritis and kidney stones.

[0005] To prevent and treat a series of diseases caused by hyperuricemia, measures need to be taken to reduce the uric acid level in the body for asymptomatic hyperuricemia patients, such as dietary control (restricting the intake of high-purine foods), increasing water intake (to promote uric acid excretion), etc. For symptomatic hyperuricemia patients, therapeutic drugs can be given. For example, the generation of uric acid in the body can be reduced by inhibiting xanthine oxidase, such as allopurinol and febuxostat; for example, promoting the excretion of uric acid with urine and reducing renal tubular reabsorption, such as probenecid, etc. In recent years, with the in-depth study of the role of gut microbiota in the degradation of uric acid, it has been found that the serum uric acid value (SUA) of hyperuricemic rats can be reduced by orally administering uricase. In addition, uricase can also be used for the treatment of tumor lysis syndrome and gout, etc., and can also be used for the preparation of blood uric acid detection kits, which is a pharmaceutical enzyme preparation with extremely wide uses. CN108103080 discloses a nucleic acid sequence encoding a recombinant uricase protein and an expression vector. After codon optimization, the nucleic acid sequence is ligated to the expression plasmid phOA, and then transformed into Escherichia coli YK537, so that the recombinant uricase exists in the periplasmic space of the cell in a soluble form, with an expression level of 20% and a cell mass yield of 80 g / L. CN114480455 discloses a functional gene fragment, recombinant strain and application for reducing blood uric acid level, which imports the uricase-encoding genes from three sources of Candida, Aspergillus niger, and Arthrobacter globiformis into the intestinal probiotic EcN through a plasmid, and can reduce uric acid from 1200 μM to 300 μM in 1 h in vitro.

[0006] Currently, uricases from a variety of different sources have been prepared or expressed, but currently, for uricases from various sources, the enzyme activity is relatively low and it is difficult to meet the requirements. Summary of the Invention

[0007] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a nucleic acid molecule encoding uricase, related products and their uses, in order to solve the above problems.

[0008] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0009] The first aspect of the present invention protects a nucleic acid molecule encoding uricase, and the nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO.2.

[0010] Among them, the uricase is derived from Alkalihalobacillus clausii; the nucleic acid molecule is a sequence after codon optimization.

[0011] The urate oxidase from different microbial sources of the present invention was codon-optimized according to the preference of Escherichia coli, aiming to further improve the expression level of uricase. A urate oxidase derived from Bacillus halodurans clausii was obtained through screening. 325 codons were adjusted, resulting in the codon adaptation index increasing from 0.48 to 0.98; the GC content increasing from 47% to 52.4%. The codon-optimized urate oxidase gene showed a higher enzyme activity after transformation, and could be expressed and correctly folded in Escherichia coli, and had a higher affinity for the substrate uric acid.

[0012] The second aspect of the present invention protects the biological materials related to the nucleic acid molecules as described above, including at least one of the following:

[0013] 1) A nucleic acid molecule encoding the urate oxidase as described above;

[0014] 2) A recombinant vector containing the nucleic acid molecule described in 1);

[0015] 3) A bioengineered bacterium containing the nucleic acid molecule described in 1), or a bioengineered bacterium containing the recombinant vector described in 2).

[0016] In certain embodiments, the protein contains the amino acid sequence shown in SEQ ID NO.3.

[0017] In certain embodiments, the recombinant vector is a prokaryotic cell recombinant vector.

[0018] In certain specific embodiments, the prokaryotic cell recombinant vector is selected from any one of the pET vector series. Preferably, the prokaryotic cell recombinant expression vector is pETDuet-1.

[0019] In certain embodiments, the bioengineered bacterium is Escherichia coli. Preferably, it is Escherichia coli (Escherichia coli BL21(DE3)).

[0020] The present invention also provides a method for constructing the bioengineered bacterium as described above. The gene encoding urate oxidase shown in SEQ ID NO.2 was synthesized by total gene synthesis and cloned into the multiple cloning site of the pETDuet-1 vector to obtain a recombinant plasmid, and then the recombinant plasmid was transformed into Escherichia coli (Escherichia coli BL21(DE3)).

[0021] The third aspect of the present invention protects a bacterial agent containing the bioengineered bacterium as described above.

[0022] In certain embodiments, the bacterial agent contains the bioengineered bacterium or its expression product or its bacterial suspension or its lysate.

[0023] The fourth aspect of the present invention protects the use of the nucleic acid molecule as described above, the biological material as described above, or the bacterial agent as described above in the preparation of urate oxidase.

[0024] The fifth aspect of the present invention protects a method for preparing urate oxidase, which uses the bioengineered bacterium or the bacterial agent as described above to prepare urate oxidase.

[0025] In some embodiments, it further includes adding an inducer.

[0026] In some specific embodiments, the inducer is selected from IPTG. Preferably, the final concentration of IPTG is 0.5 - 1.5 mmol / L.

[0027] In some embodiments, it further includes post-treatment, and the post-treatment includes one or more of solid-liquid separation, homogenization, and purification of the cultured product.

[0028] Preferably, the solid-liquid separation is centrifugation. The speed and time of centrifugation can be screened by those skilled in the art according to actual needs, such as it can be at 4°C, 8000 rpm, 12000 rpm, and the time is 5 - 15 min.

[0029] Preferably, the pressure of homogenization is 700 - 900 bar.

[0030] More preferably, a cell disruption solution is added before homogenization to keep the released urate oxidase at a relatively high activity. The composition of the cell disruption solution is 20 mM Tris-HCL, pH 7.4, 300 mM NaCl, 1 mM PMSF, 1% Triton x-100, 1 mM β-mercaptoethanol. The addition amount of the cell disruption solution is 10 - 30 v / v% of the volume of the cells after solid-liquid separation.

[0031] In some embodiments, the bioengineered bacterium is cultured to obtain a seed solution in an LB liquid medium containing 100 μg - 1 mg / mL ampicillin, and the seed solution is inoculated into a TB fermentation medium containing 100 μg - 1 mg / mL ampicillin for culture. When the OD 600 is 0.4 - 0.6, an inducer is added for induction.

[0032] In some specific embodiments, the culture temperature is 20 - 45°C.

[0033] In some specific embodiments, the induction is carried out at 15 - 37°C.

[0034] In certain specific embodiments, the LB (Luria broth) liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, adjust the pH to 7.4 with 1 mL of 1 mol / L NaOH, make up the volume to 1 L with deionized water, and sterilize by autoclaving at 121 °C for 20 min.

[0035] In certain specific embodiments, the TB liquid fermentation medium: 11.8 g of tryptone, 23.6 g of yeast extract, dissolve in 900 mL of deionized water. Add 5 mL of 80% glycerol (pre-prepared). Sterilize by autoclaving at 115 °C for 20 minutes. Prepare the phosphate solution: Weigh 9.4 g of K2HPO4 and 2.2 g of KH2PO4 on the balance, dissolve in 100 mL of deionized water. Sterilize by autoclaving at 115 °C for 20 minutes, or filter-sterilize with 0.2 μm. When the medium cools to below 60 °C, pour in the phosphate solution.

[0036] The sixth aspect of the present invention protects the uricase obtained by the preparation method as described above.

[0037] The enzyme activity of the uricase of the present invention derived from Alkalihalobacillus clausii can reach 249.19 IU / mL, which is far higher than that of uricase from other microbial sources. The catalytic pH value is 7-11 and the enzyme activity increases with the increase of pH value; the catalytic temperature is 30-60 °C, and it can still survive at 60 °C and is thermally stable.

[0038] The seventh aspect of the present invention protects the use of the nucleic acid molecule as described above, the biological material as described above, the bacterial agent as described above, or the uricase as described above in at least one of the following:

[0039] 1) Reducing purines in food;

[0040] 2) Preparing a product for reducing uric acid;

[0041] 3) Preparing a detection kit;

[0042] 4) Preparing a drug or health product for reducing uric acid.

[0043] The present invention uses the obtained uricase to enzymatically hydrolyze food, which can reduce the levels of purines and their oxidation product uric acid in food, and has a good effect on preventing gout. The uricase of the present invention enzymatically hydrolyzes the mixture of soy milk and xanthine oxidase, and can reduce the uric acid content from 1.07 mg / L to 0 mg / L, indicating that the uricase of the present invention can significantly reduce the levels of purines and their oxidation product uric acid in food. The food includes soy milk, high soup, beer, etc.

[0044] The urate oxidase or biological material of the present invention can be used as a raw material to prepare products for reducing uric acid to lower uric acid, and the products can be bacterial agents, enzyme preparations, additives, or foods.

[0045] The urate oxidase or biological material of the present invention can be used to prepare a detection kit. The principle is that urate oxidase can catalyze uric acid to generate hydrogen peroxide and allantoin. The content of uric acid in the sample to be detected can be detected by detecting the quinone red compound produced by the reaction of hydrogen peroxide with 4 - aminophenazone and phenol.

[0046] The urate oxidase or biological material of the present invention can be used to prepare drugs or health products for reducing uric acid levels to prevent or treat hyperuricemia.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1) The present invention first obtained the urate oxidase gene sequence from Alkalihalobacillus clausii for expression in Escherichia coli, which has low cost, simple molecular operation, is easy to clone and express, and has good repeatability.

[0049] 2) The present invention constructed a recombinant vector and a bioengineered bacterium for urate oxidase expression, and successfully obtained an enzyme protein with a molecular weight of 35 kDa. It has high protease activity and is simple to prepare.

[0050] 3) The urate oxidase of the present invention can be used in the production of low - purine foods to reduce the purine content in foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It shows the electrophoresis pattern of the crude enzyme solution of urate oxidase from different microbial sources expressed in host cells in Example 2 of the present invention.

[0052] Figure 2 It shows the enzyme activity results of the crude enzyme solution of urate oxidase from different microbial sources expressed in host cells in Example 3 of the present invention.

[0053] Figure 3 It shows the results of the effects of different pH values on the enzyme activity and stability of urate oxidase in Example 3 of the present invention.

[0054] Figure 4 It shows the results of the effects of different temperatures on the enzyme activity and stability of urate oxidase in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0055] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0056] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0057] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0058] The culture media used in the following embodiments of this application are as follows:

[0059] LB (Luria broth) liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, adjust the pH to 7.4 with 1 mL of 1 mol / L NaOH, make up the volume to 1 L with deionized water, and sterilize by high-pressure steam at 121 °C for 20 min.

[0060] LB solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 20 g of agar powder, adjust the pH to 7.4 with 1 mL of 1 mol / L NaOH, make up the volume to 1 L with deionized water, and sterilize by high-pressure steam at 121 °C for 20 min.

[0061] TB liquid fermentation medium: Weigh 11.8 g of tryptone and 23.6 g of yeast extract on a balance, dissolve them in 900 mL of deionized water. Add 5 mL of 80% glycerol (pre-prepared). Sterilize by high-pressure steam at 115 °C for 20 minutes. Prepare the phosphate solution: Weigh 9.4 g of K2HPO4 and 2.2 g of KH2PO4 on a balance, dissolve them in 100 mL of deionized water. Sterilize by high-pressure steam at 115 °C for 20 minutes or filter-sterilize with 0.2 μm. When the medium cools to below 60 °C, pour in the phosphate solution.

[0062] The reagents, enzymes and related kits used in the following embodiments of this application are as follows:

[0063] Ampicillin was purchased from Sangon Biotech Co., Ltd. (Shanghai). All chemical reagents are of analytical grade and were purchased from Sangon Biotech Co., Ltd. (Shanghai). All restriction endonucleases and DNA ligases were purchased from Thermo. The 1kb DNA Ladder was purchased from Takara. The plasmid miniprep kit and gel extraction kit were purchased from Sangon Biotech Co., Ltd. (Shanghai).

[0064] The strains and plasmids used in the following embodiments of this application are as follows:

[0065] Escherichia coli BL21(DE3) was used for the expression of recombinant vectors and strain fermentation. The plasmid pETDuet-1 was purchased from Novagen (Darmstadt, Germany).

[0066] The strains and plasmids used in the following embodiments of this application are shown in Table 1 below:

[0067] Table 1

[0068] Instrument Manufacturer Bench-top high-speed centrifuge Shanghai Lichen Technology 64RL high-speed refrigerated centrifuge Luxe Instrument Co., Ltd. Constant temperature oscillator Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Constant temperature incubator Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. LDZX-40AI vertical electric heating pressure sterilizer Shanghai Shen'an Medical Device Factory Visible light spectrophotometer Shanghai Metash Instruments Co., Ltd. Gel imaging system UVP, USA DYY-6C electrophoresis apparatus Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd. Hot air blast drying oven Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Ultra-low temperature refrigerator Meiling PCR instrument BIO-RAD, USA pH meter Mettler-Toledo, Switzerland Rotary evaporator dryer Heidolph, Germany HPLC Shimadzu, Japan Digital display water bath Shanghai Yiheng Technology Co., Ltd.

[0069] Example 1 Codon Optimization and Recombinant Vectors

[0070] In this example, urate oxidases from different sources were codon-optimized and then ligated to the plasmid pETDuet-1 respectively to obtain recombinant vectors. The details are as follows:

[0071] 1.1 Screening of Urate Oxidases from Different Sources

[0072] According to the urate oxidase gene sequences from different sources provided by NCBI, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the full gene sequences. The urate oxidase genes from different sources are shown in Table 1.

[0073] Table 1

[0074]

[0075]

[0076] 1.2 Codon Optimization

[0077] Based on the codon preference of the E. coli expression system, factors such as GC content, codon balance, mRNA secondary structure, nuclease cleavage sites, trans-acting element binding, and restriction site avoidance were comprehensively evaluated, and the full-length nucleotide sequences of urate oxidase genes from different sources were optimized.

[0078] The gene (Gene ID: 61573289) of urate oxidase derived from Alkalihalobacillus clausii is shown as SEQ ID NO.1.

[0079] The nucleotide sequence after codon optimization is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO.3.

[0080] This optimization involved a total of 325 amino acid codon adjustments. In the optimization, in addition to changing some rare codons to preferred codons according to the preference of Escherichia coli to improve the adaptation index of the target gene in the host, the codon adaptation index was increased from 0.48 to 0.98; at the same time, the GC content of the entire sequence was adjusted from 47% to 52.4%, enhancing the stability of the urate oxidase coding sequence and benefiting the positive regulation of the efficiency of mRNA translating proteins.

[0081] SEQ ID NO1

[0082] gtggaaaataaacggacaatgtcttacggaaaagggaatgtgtttgcctaccgaacgtttatggaaccgttaagcgggcttcagccgatccctgaatcggcatttactgttcgggataacactgtttttggcatcaatgtcacagttgaagtcggggggaacgcttttctgtcgtcttttacagagggggacaatcaaatggttgttgcaaccgattctatgaaaaactttatccaacggcatttagcgacattttcaggccggaccattgagggctttatccgttatgtcggggaggcttttctaacgacttacagccacatagactgggtcaagctaacaggcgaagccgtgccttttgaaaatactacgtatgcgaatggggaagaaccaagcacaagtacactcgtttataagcactcgcgcaatgaacgaaacgaagcgtcgattgagctcgttcgcgaaggaaacggctggcgaataaaccgccagaacagcgcactccttgacttgcagcttgttaaagtgaaggacaattcatttgttggctttattcgcgatcaatatacaacgctgcctgaggattccaatcgaccgcttttcatttacttgaacattggctggtcttacgaaacagacgacgatgcgctaggggaggaacctccccgttatgtggctggagaacaagttgccgatctcgcttcttctgtttttcatgaactggcctccccctcgattcaacacttggtttaccaaatcggctgtcgcatgttaaaacgatttccccagttgcaagaagtaacgtttgaatcgcaaaaccgtacttgggacacagttgtagaagaccttcccgaaacagaaggaaaagtgtatacggagccgcgcttgccatttggctttcaacggttttccgttacaaaagcagatctcgccactcaaaccacgtccaatagaactgagacaacgcgattatga

[0083] SEQ ID NO2

[0084] atggtagaaaacaaacgtaccatgagctacggcaaaggtaacgttttcgcgtaccgtaccttcatggaaccgctgagcggcctgcagccgatcccggaatctgctttcaccgttcgtgataacaccgtttttggtattaacgttaccgttgaagttggtggcaacgcattcctgtctagcttcaccgaaggcgataaccagatggttgttgctaccgattctatgaaaaacttcatccagcgtcacctggcgaccttcagcggccgtaccatcgaaggtttcatccgttacgttggcgaagcgttcctgaccacctactctcacattgattgggttaaactgaccggtgaagcagttccgttcgaaaacaccacctacgctaacggcgaagaaccgagcacctctaccctggtttataaacacagccgtaacgaacgtaacgaagcgagcatcgaactggttcgtgaaggtaacggttggcgtatcaaccgtcagaacagcgcgctgctggatctgcagctggttaaagttaaagataacagcttcgttggtttcatccgtgaccagtacaccaccctgccggaagatagcaaccgtccgctgttcatctacctgaacatcggttggagctatgaaaccgatgatgatgcgctgggtgaagaaccgccgcgttacgttgcgggcgaacaggttgcggatctggcgagctctgtgttccacgaactggcttccccgtctatccagcacctggtttaccagatcggctgccgtatgctgaaacgtttcccgcagctgcaggaagttaccttcgaatctcagaaccgtacctgggataccgttgttgaagatctgccggaaaccgaaggtaaagtttataccgaaccgcgtctgccgttcggtttccagcgtttctctgttaccaaagcggatctggcgacccagaccaccagcaaccgtaccgaaaccacccgtctgtaa

[0085] SEQ ID NO3

[0086] VENKRTMSYGKGNVFAYRTFMEPLSGLQPIPESAFTVRDNTVFGINVTVEVGGNAFLSSFTEGDNQMVVATDSMKNFIQRHLATFSGRTIEGFIRYVGEAFLTTYSHIDWVKLTGEAVPFENTTYANGEEPSTSTLVYKHSRNERNEASIELVREGNGWRINRQNSALLDLQLVKVKDNSFVGFIRDQYTTLPEDSNRPLFIYLNIGWSYETDDDALGEEPPRYVAGEQVADLASSVFHELASPSIQHLVYQIGCRMLKRFPQLQEVTFESQNRTWDTVVEDLPETEGKVYTEPRLPFGFQRFSVTKADLATQTTSNRTETTRL

[0087] The gene of urate oxidase derived from Bacillus subtilis is shown as SEQ ID NO.4.

[0088] The nucleotide sequence after codon optimization is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6.

[0089] The gene of urate oxidase derived from Brevibacterium casei is shown as SEQ ID NO.7.

[0090] The nucleotide sequence after codon optimization is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO.9.

[0091] The gene of urate oxidase derived from Neurospora crassa is shown as SEQ ID NO.10.

[0092] The nucleotide sequence after codon optimization is shown as SEQ ID NO.11, and the amino acid sequence is shown as SEQ ID NO.12.

[0093] The gene of urate oxidase derived from Streptomyces coelicolor is shown as SEQ IDNO.13.

[0094] The nucleotide sequence after codon optimization is shown as SEQ ID NO.14, and the amino acid sequence is shown as SEQ ID NO.15.

[0095] The codon-optimized urate oxidase gene sequences from different sources were sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis, and the restriction enzyme cleavage sites shown in Table 1 were added at both ends of the sequences to obtain 5 urate oxidase fragments for subsequent vector construction.

[0096] 1.3 Construction of recombinant vectors

[0097] The urate oxidase fragments obtained in step 1.1 were digested with NdeI and KpnI, and then ligated to the plasmid pETDuet-1 digested with NdeI and KpnI. The ligation was carried out according to the instruction manual of the restriction endonuclease and ligase kit from Thermo.

[0098] Five recombinant vectors, pET-AC-pucL, pET-BS-PUL, pET-BC-UCL, pET-NC-URI, and pET-SC-URI, were obtained.

[0099] Example 2 Construction and induced expression of engineering bacteria

[0100] The recombinant vectors constructed in Example 1 were respectively transformed into Escherichia coli competent cells for protein expression.

[0101] 2.1 Transformation into Escherichia coli competent cells

[0102] 2.1.1 Preparation of Escherichia coli (Escherichia coli BL21(DE3)) competent cells

[0103] 1) Pick a single colony of Escherichia coli (Escherichia coli BL21(DE3)) into 5 mL of LB liquid medium and culture it overnight at 37 °C and 200 rmp on a shaker for about 12 h.

[0104] 2) Transfer 500 μL of the overnight culture to 50 mL of fresh LB liquid medium and culture it at 37 °C and 200 rmp on a shaker until the absorbance reaches 0.35 - 0.6 at 600 nm to obtain a bacterial solution.

[0105] 3) Pre-cool 0.1 mol / L CaCl2 solution on ice.

[0106] 4) Pipette 25 mL of the bacterial solution obtained in step 2) into a 50 mL EP tube, cool it on ice for 10 min. Centrifuge it at 3000 g, 5 min and 4 °C in a high-speed refrigerated centrifuge, discard the supernatant, add 1.666 mL of the pre-cooled 0.1 mol / L CaCl2 solution from step 3), gently pipette up and down with a pipette to resuspend the cells, and place it on ice for 20 min.

[0107] 5) Centrifuge at 3000 g for 5 min at 4 °C in a high-speed refrigerated centrifuge, discard the supernatant, add 1 mL of pre-cooled mixed solution (the pre-cooled mixed solution contains 0.1 mol / L CaCl2 and 10 wt% glycerol), pipette and mix well to resuspend the cells to obtain a cell suspension.

[0108] 6) Aliquot 100 μL of the cell suspension into 1.5 mL EP tubes, store at -70 °C for later use to obtain Escherichia coli competent cells.

[0109] 2.1.2. Transformation into Escherichia coli competent cells using the heat shock method

[0110] 1) After the Escherichia coli competent cells are thawed, transfer 1 μL of each of the 5 recombinant vectors constructed in Example 1 into the EP tubes containing 100 μL of competent cells, and gently flick to mix well.

[0111] 2) Place on ice for 30 min, then perform heat shock in a 42 °C water bath for 90 s, and place on ice for 10 min.

[0112] 3) Add 1 mL of fresh LB liquid medium to the EP tubes after heat shock transformation, and place in a shaker for culture (37 °C, 200 rpm, 1 h). Add all the Escherichia coli to the corresponding resistance plates of the plasmid (i.e., containing 0.1 mg / mL ampicillin), and spread evenly.

[0113] 4) Incubate the transformation plates upside down in a 37 °C incubator for 12 - 24 h to obtain the engineered bacteria after transformation, which are respectively labeled as BL21(E-AC-pucL), BL21(E-BS-PUL), BL21(E-BC-UCL), BL21(E-NC-URI), BL21(E-SC-URI).

[0114] 2.2. Induced expression

[0115] The engineered bacteria after transformation are first cultured overnight in LB liquid medium containing 0.1 mg / mL ampicillin at 37 °C and 200 rpm in a shaker to obtain a seed culture, and then re-inoculated into TB fermentation medium containing 0.1 mg / mL ampicillin at an initial inoculum of OD 600 = 0.1 (i.e., when inoculating the seed culture into the TB fermentation medium, measure the OD of the mixed product 600 = 0.1), and culture in a shaker at 37 °C and 200 rpm until OD 600 = 0.5.

[0116] Then add IPTG (Isopropyl-β-D-thiogalactopyranoside) with a final concentration of 1 mmol / L for induction, and culture and ferment in a shaker at 20 °C and 200 rpm for 20 h.

[0117] 2.3, Centrifugal harvesting of crude enzyme solution

[0118] 1) Centrifugally collect the bacterial cells: Centrifuge the fermentation product after the fermentation in step 2.2 at 4 °C and 8000 rpm for 10 minutes. After discarding the supernatant, resuspend and wash the bacterial cells three times with a resuspension solution. The resuspension solution is Tris-HCl with a pH value of 7.5.

[0119] 2) High-pressure homogenization for disruption: Resuspend the bacterial cells again with the cell disruption solution and adjust the concentration of the bacterial cells to 20 v / v%. The temperature for high-pressure homogenization is 4 °C, the disruption pressure is 800 bar, and it cycles twice.

[0120] 3) Centrifuge to obtain the crude enzyme solution: After disruption, centrifuge at 4 °C and 12000 rpm for 10 minutes to collect the supernatant, obtaining the crude enzyme solution.

[0121] The composition of the cell disruption solution is: 20 mM Tris-HCL, pH value of 7.4, 300 mM NaCl, 1 mM PMSF, 1% Tris-x100, 1 mM β-mercaptoethanol.

[0122] 2.4, SDS-PAGE gel electrophoresis

[0123] Treat the sample: Take 160 μL of the crude enzyme solution obtained in step 2.3, add 40 μL of 5X loading buffer, mix well, place it in a 100 °C water bath and boil for 5 min. Place the pretreated sample at 4 °C for standby.

[0124] Perform SDS-PAGE gel electrophoresis on the treated sample. The operation of gel electrophoresis is as follows:

[0125] Perform discontinuous vertical slab electrophoresis analysis using a 12% separating gel and a 4% stacking gel. After turning on the power supply, maintain a constant voltage of 60 V until the sample enters the separating gel from the stacking gel stage, then adjust the voltage to 120 V and maintain it until the bromophenol blue moves to about 0.5 cm from the bottom of the gel and stop electrophoresis. Transfer the gel to Coomassie Brilliant Blue staining solution for staining for 2 h, and then decolorize it thoroughly with the decolorizing solution for 10 h. Place the decolorized gel under the white light condition of a gel imager to observe the protein subunit bands. The results are shown in Figure 1 . Among them, bands 1, 2, 3, 4, and 5 are the engineered bacteria after modification, namely E-AC-pucL, E-BS-PUL, E-BC-UCL, E-NC-URI, E-SC-URI; bands 6 and 7 are pETDuet-1 without IPTG induction and pETDuet-1 with IPTG induction.

[0126] As Figure 1As shown, protein electrophoresis can detect a protein band of 35 kDa, and the size conforms to the expected design; while no protein was expressed in pETDuet-1 with or without IPTG added.

[0127] Example 3 Determination of Enzymatic Properties of Urate Oxidase

[0128] The crude enzyme solution obtained in Example 2 was subjected to enzyme activity determination, including the following:

[0129] 3.1 Determination of Urate Oxidase Activity

[0130] The crude enzyme solution was diluted and then subjected to enzyme activity determination. Specifically as follows:

[0131] Prepare the reaction solution: 200 μL of 1200 μmol / L uric acid, 1.8 mL of 50 mmol / L KH2PO4-K2HPO4 buffer solution, and the pH of the buffer solution is 8.0.

[0132] Take 2 mL of the reaction solution and preheat it in a 60 °C water bath for 5 min. Then add 20 μL of the crude enzyme solution produced by the 5 modified engineering bacteria obtained in Example 2 and catalyze the reaction at 37 °C for 5 min. After the reaction, add 200 μL of 20 wt% KOH aqueous solution to inactivate the enzyme, and measure the absorbance at a wavelength of 293 nm. The enzyme activity unit was calculated according to the decrease value of uric acid in the reaction system. During the determination, dilute the crude enzyme solution to an appropriate multiple as needed.

[0133] Definition of activity unit: The amount of enzyme required to catalyze the decomposition of 1 μmol of uric acid per minute under this condition is 1 unit (U).

[0134] The enzyme activity calculation formula is: U / mL = (△A * VT * df) / (12.2 * VE * t)

[0135] In the formula, U / mL is the number of enzyme activity units per mL of urate oxidase; ΔA is the decrease value of absorbance at a wavelength of 293 nm during the reaction for 5 min at 37 °C; VT is the total volume of the reaction solution (mL) (i.e., the volume of the reaction solution + the volume of the crude enzyme solution); df is the dilution multiple; 12.2 is the micromolar extinction coefficient of uric acid at a wavelength of 293 nm; VE is the sample volume (i.e., the volume of the crude enzyme solution); t is the reaction time. The results of enzyme activity determination are shown in Figure 2 .

[0136] From Figure 2 it can be seen that the enzyme activity of the crude fermentation enzyme solution expressed by E-AC-pucL is the best, reaching 249.19 IU / mL, while the enzyme activities of the crude enzyme solutions produced by other engineering bacteria are only less than 40 IU / mL. Currently, the reported enzyme activity of urate oxidase in the literature is only 67 IU / mL. Therefore, the crude enzyme solution ac-pucL produced by the E-AC-pucL engineering bacteria was used for subsequent research.

[0137] 3.2, Effect of Different pH Values on Uricase

[0138] Determination of the Optimal pH Value: Replace the same volume of KH2PO4-K2HPO4 buffer solution in Step 3.1 with buffer solutions of different pH values (pH 4.0 - 10.0). The concentrations of uric acid and crude enzyme solution are the same. Determine the enzyme activity according to the method in Step 3.1. Take the one with the highest enzyme activity as 100%, obtain the relative enzyme activity, and determine the optimal reaction pH of uricase based on the relative enzyme activity.

[0139] The buffer solutions of different pH values are as follows:

[0140] Buffer solution with pH 4.0 - 5.0: 50 mM sodium acetate.

[0141] Buffer solution with pH 5.5 - 8.0: 50 mM sodium phosphate.

[0142] Buffer solution with pH 8.5 - 9.5: 50 mM Tris-HCl.

[0143] Buffer solution with pH 10.0 - 11.0: 50 mM sodium carbonate.

[0144] Investigation of pH Stability: Dilute the crude enzyme solution ac-pucL with buffer solutions of different pH values (pH 4.0 - 11.0), without adding uric acid. After incubating at 25°C for 16 h, measure the remaining enzyme activity.

[0145] Take the one with the highest enzyme activity as 100%, and the ratio of the remaining enzyme activity to it is the relative enzyme activity. Make a curve of relative enzyme activity against pH value to investigate the pH stability of uricase. The results are shown in Figure 3 .

[0146] As can be seen from Figure 3 a, the enzyme activity of uricase ac-pucL derived from Lactobacillus alkalophilus Kraus gradually increases with the increase of pH, reaches the highest at pH 8.06, and maintains a relatively high activity under alkaline conditions.

[0147] As can be seen from Figure 3 b, the uricase ac-pucL derived from Lactobacillus alkalophilus Kraus has the best stability at pH 7.5.

[0148] 3.3, Effect of Different Temperatures on Uricase

[0149] Optimal reaction temperature: The enzyme activity was measured according to the method of step 3.1 at catalytic reaction temperatures of 30°C, 37°C, 50°C, 60°C, 70°C, and 80°C respectively in a buffer solution (50 mM sodium phosphate buffer) with a pH of 7.5. Taking the highest enzyme activity as 100%, the optimal reaction temperature of urate oxidase was determined. The results are shown in Figure 4 a.

[0150] Thermostability investigation: The crude urate oxidase solution ac-pucL was incubated at 20°C, 30°C, 37°C, 50°C, 60°C, 70°C, and 80°C for 30 min respectively, then quickly cooled to 25°C, and the remaining enzyme activity was measured without adding uric acid. Taking the enzyme activity measured from the crude enzyme solution stored at 4°C without incubation as 100%, the relative enzyme activity was calculated, and a temperature-relative enzyme activity curve was made to investigate the thermostability of urate oxidase. The results are shown in Figure 4 b.

[0151] From Figure 4 a, it can be seen that the urate oxidase ac-pucL derived from Lactobacillus alkaliphilus Krausii has the highest enzyme activity at 37°C, and the enzyme activity gradually decreases with the increase of temperature.

[0152] From Figure 4 b, it can be seen that the urate oxidase ac-pucL derived from Lactobacillus alkaliphilus Krausii can survive well at 20 - 80°C.

[0153] In summary, the catalytic temperature of urate oxidase is 30 - 60°C, and the optimal catalytic temperature is 37°C; the catalytic pH value is 7 - 11, and the optimal catalytic pH value is 8.06. At the same time, the urate oxidase has good pH stability, maintaining a high activity between pH 6 - 9, and the remaining enzyme activity is above 70%. In addition, the urate oxidase has good thermostability. After incubation at 20°C, 37°C, and 50°C for 30 min, the remaining enzyme activity is all above 70%; after incubation at 60°C, 70°C, and 80°C for 30 min, the remaining enzyme activity is all above 20%.

[0154] Example 5 Application of urate oxidase in reducing uric acid in food

[0155] In this example, by adding commercially available xanthine oxidase to soy milk, hypoxanthine in soy milk was converted into xanthine, and at the same time xanthine was converted into uric acid. Then, the crude enzyme solution ac-pucL obtained in Example 2 was added to the reaction system for evaluation.

[0156] The xanthine oxidase was purchased from Hubei Jianchu Biotechnology Co., Ltd.

[0157] System No. 1 consisted of 2 mL of soy milk and 20 U of xanthine oxidase.

[0158] The No. 2 system consists of 2 mL of soymilk, 20 U of xanthine oxidase, and 10 μL of crude uricase solution ac-pucL.

[0159] After incubation at 40 °C for 2 h, it was inactivated by boiling at 100 °C for 10 min, and the reaction was terminated by inactivating the enzyme. After the reaction was completed, the uric acid content in the reaction solution was detected.

[0160] Detection of uric acid content: The components of the reaction mixture were detected by HPLC method. After filtering through a 0.22 μm syringe filter, 10 μL of the reaction solution was injected into a ZORBAX SB-Aq C18 column (L.N. B15072, Agilent), and the ultraviolet absorption spectrum at 254 nm was collected. HPLC analysis was carried out at 40 °C with a flow rate of 1 mL / min on a Shimadzu HPLC system equipped with a 2998 photodiode array detector.

[0161] The mobile phase was: 0.02 mol / l potassium dihydrogen phosphate, and the pH was adjusted to 2.8 with phosphoric acid. The standard sample of uric acid was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0162] After detection, in the No. 1 system after adding xanthine oxidase, the uric acid concentration increased to 1.07 mg / L. After treatment with the crude uricase solution of Lactobacillus kefiranofaciens, the uric acid content in the soymilk of the No. 2 system decreased to 0 mg / L, indicating that the uricase of the present invention can be applied to the degradation of uric acid in food systems.

[0163] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A nucleic acid molecule encoding urate oxidase, characterized in that, The nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.

2.

2. A biological material related to the nucleic acid molecule as described in claim 1, characterized in that, It includes at least one of the following: 1) A protein encoded by the nucleic acid molecule as described in claim 1; 2) A recombinant vector containing the nucleic acid molecule as described in claim 1; 3) A genetically engineered bacterium containing the nucleic acid molecule as described in claim 1, or a genetically engineered bacterium containing the recombinant vector described in 2).

3. The biomaterial according to claim 2, wherein The protein comprises the amino acid sequence shown in SEQ ID NO.3; and / or, the recombinant vector is a prokaryotic cell recombinant vector; and / or, the genetically engineered bacterium is Escherichia coli.

4. The biomaterial according to claim 3, characterized in that, The prokaryotic cell recombinant vector is selected from any one of the pET vector series; preferably, the prokaryotic cell recombinant expression vector is pETDuet-1.

5. A bacterial agent, characterized in that, It contains the genetically engineered bacterium as described in claim 2.

6. Use of the nucleic acid molecule as described in claim 1, the biological material as described in any one of claims 2-4, or the bacterial agent as described in claim 5 in the preparation of urate oxidase.

7. A method for preparing urate oxidase, characterized in that, Prepare urate oxidase using the genetically engineered bacterium as described in claim 2 or the bacterial agent as described in claim 5.

8. The preparation method according to claim 7, characterized in that, It also includes adding an inducer; preferably, the inducer is selected from IPTG.

9. Urate oxidase obtained by the preparation method as described in any one of claims 7-8.

10. Use of the nucleic acid molecule as described in claim 1, the biological material as described in any one of claims 2-4, the bacterial agent as described in claim 5, or the urate oxidase as described in claim 9 in at least one of the following: 1) Reducing purine in food; 2) Preparing a product for degrading uric acid; 3) Preparing a detection kit; 4) Preparing a drug or health product for reducing uric acid.

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