Black yeast uricase and its mutant and application
The uric acid oxidase sequence is obtained through deep learning and the highly active mutants are obtained through semi-rational design and transformation technology, which solves the problems of insufficient uric acid oxidase yield and cumbersome purification process, achieves efficient expression and reduces costs, and has great application potential.
Patent Information
- Application Number
- CN202410655008.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-24
- Publication Date
- 2025-05-16
- Estimated Expiration
- 2044-05-24
AI Technical Summary
In the prior art, the insufficient production of uric acid oxidase and the cumbersome purification process lead to high costs and are not widely used in the treatment of hyperuricemia.
The uric acid oxidase sequence was obtained through deep learning, and the gene encoding uric acid oxidase had uric acid oxidase activity, and a uric acid oxidase mutant with high activity was obtained through semi-rational design and modification technology. This mutant was used to express uric acid oxidase to degrade uric acid.
It improves the specific enzyme activity of uric acid oxidase, achieves efficient expression in E. coli, reduces production costs, and has great application potential.
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Figure CN118497162B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of enzyme engineering, and in particular relates to a black yeast urate oxidase and a mutant and application thereof. Background Art
[0002] Hyperuricemia is a common metabolic disease, which is associated with a variety of diseases of the cardiovascular and metabolic systems, such as hypertension and coronary artery disease. Currently, the treatment drugs for hyperuricemia include allopurinol, febuxostat, benzbromarone, probenecid, etc. Although they all have the effect of degrading uric acid, long-term use will cause adverse reactions such as rash allergies, liver damage, and kidney stones. One-third of human uric acid is excreted by the intestines, and two-thirds is excreted by the kidneys. During the evolutionary process, human urate oxidase lost its activity due to gene mutation. This means that under the condition of a high-purine diet, human uric acid metabolism may be abnormal, leading to uric acid accumulation, which may cause gout and hyperuricemia. Urate oxidase, as an enzyme that can directly catalyze the degradation of uric acid, has great potential in the treatment of hyperuricemia. However, due to the insufficient production of urate oxidase and the cumbersome purification process resulting in excessively high costs, it has not been widely used in China. Therefore, it is of great significance to construct a genetically engineered strain that produces high urate oxidase. Summary of the invention
[0003] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a black yeast urate oxidase and a mutant and application thereof.
[0004] The present invention obtains the uricase sequence based on deep learning, thereby verifying for the first time that the gene encoding uricase has uricase activity. Furthermore, amino acid mutation points are obtained through semi-rational design and transformation technology, and uricase oxidase mutant engineered bacteria with higher activity are obtained. The strain is used to express uricase oxidase to achieve the effect of degrading uric acid.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] A black yeast urate oxidase HorUox mutant, whose amino acid sequence is SEQ ID NO.2 obtained by any one of the following mutations:
[0007] (1) E183Y, i.e. the amino acid at position 183 mutates from E to Y, and the same applies to the others;
[0008] (2) L287Y,
[0009] (3)D283R.
[0010] A gene encoding a mutant as described above.
[0011] The mutant-related biological material is any one or more combinations of the following biological materials:
[0012] (a) an expression cassette containing the above encoding gene;
[0013] (b) a recombinant expression vector containing the above encoding gene;
[0014] (c) a recombinant expression vector containing the expression cassette described in (a);
[0015] (d) a recombinant microorganism containing the above encoding gene;
[0016] (e) a recombinant microorganism containing the expression cassette described in (a);
[0017] (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).
[0018] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a pET series vector, etc., preferably a pET28a(+) vector.
[0019] Furthermore, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes, etc. The prokaryotes include bacteria such as Escherichia. More specifically, the prokaryotes are Escherichia coli (E. coli), and specifically can be Escherichia coli BL21 (DE3).
[0020] The mutant, the encoding gene and the mutant-related biological materials are used in preparing the black yeast urate oxidase HorUox mutant.
[0021] Furthermore, the mutants, encoding genes, mutant-related biomaterials, black yeast urate oxidase HorUox, and black yeast urate oxidase HorUox-related biomaterials are used in the preparation of products for degrading uric acid.
[0022] The biological material related to the black yeast urate oxidase HorUox is any one or more combinations of the following biological materials:
[0023] 1) an expression cassette containing a gene encoding the black yeast urate oxidase HorUox;
[0024] 2) a recombinant expression vector containing a gene encoding black yeast urate oxidase HorUox;
[0025] 3) a recombinant expression vector containing the expression cassette described in 1);
[0026] 4) A recombinant microorganism containing a gene encoding HorUox, a urate oxidase from black yeast;
[0027] 5) A recombinant microorganism containing the expression cassette described in 1);
[0028] 6) A recombinant microorganism containing the recombinant expression vector described in 2) or 3).
[0029] Furthermore, the starting vector of the recombinant expression vector in 2) and 3) is a pET series vector, etc.; preferably, it is a pET28a(+) vector.
[0030] Furthermore, the host microorganism corresponding to the recombinant microorganism in 4), 5), and 6) is selected from prokaryotes, etc.; the prokaryotes include bacteria such as Escherichia. More specifically, the prokaryotes are Escherichia coli (E. coli), and specifically can be Escherichia coli BL21 (DE3).
[0031] Further preferably, the recombinant microorganisms described in (d), (e), (f), 4), 5), and 6) also contain the 5-hydroxyisouric acid hydrolase gene pucM to achieve the dual enzyme cascade expression of HorUox and pucM, thereby realizing the process from uric acid to allantoin via 5-hydroxyisouric acid.
[0032] Among them, the amino acid sequence of the black yeast urate oxidase HorUox is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the black yeast urate oxidase HorUox is shown in SEQ ID NO.1.
[0033] The 5-hydroxyisouridate hydrolase is derived from Cyberlindnera jadinii; the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
[0034] A method for obtaining the above mutant comprises the following steps: performing site-directed mutagenesis on a gene encoding a black yeast urate oxidase HorUox having an amino acid sequence as shown in SEQ ID NO.2 by designing primers containing mutation sites, and then expressing the resultant to obtain a black yeast urate oxidase HorUox mutant.
[0035] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding the black yeast urate oxidase HorUox having an amino acid sequence as shown in SEQ ID NO.2. After correct sequencing, the gene was transformed into Escherichia coli BL21 (DE3) for expression to obtain a black yeast urate oxidase HorUox mutant.
[0036] Compared with the prior art, the present invention has the following advantages and effects:
[0037] (1) The black yeast urate oxidase HorUox provided by the present invention is obtained by deep learning, a large number of enzyme activity calculations combined with phylogenetic tree screening, and is optimized and modified by the codon preference of Escherichia coli, which is conducive to its expression in Escherichia coli.
[0038] (2) The present invention utilizes a prokaryotic expression system and selects pET28a(+) vector to construct a recombinant plasmid and transfer it into Escherichia coli BL21(DE3). After low-temperature induced fermentation culture, the recombinant uricase oxidase is expressed in a soluble manner. The nucleotide sequence encoding uricase oxidase was transformed through molecular docking, semi-rational design, molecular dynamics simulation, etc. to obtain mutants HorUm(E183Y), HorUm(D283R), and HorUm(L287Y). The specific enzyme activities of the mutants were measured to be 2.02 times, 1.35 times, and 1.73 times higher than those of the wild type, respectively, and have great application potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 This is a schematic diagram of the construction of the expression vector pET28a(+)-HorUm.
[0040] Figure 2 It is the uric acid standard curve; wherein, the horizontal axis is the uric acid (ie UA) concentration μM, and the vertical axis is the HPLC peak area (microvolt*second).
[0041] Figure 3 is the uric acid standard curve; the horizontal axis is the urea concentration (μmol / L), and the vertical axis is the absorbance value at 293nm (OD 293 ).
[0042] Figure 4 This is a graph showing the results of determining the optimal reaction pH of recombinant urate oxidase.
[0043] Figure 5 This is a graph showing the results of determining the optimal reaction temperature of recombinant urate oxidase. DETAILED DESCRIPTION
[0044] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental operations involved in the following implementation cases are well known to those skilled in the art.
[0045] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.
[0046] The main experimental materials and sources used in the implementation case are as follows:
[0047] (1) Experimental materials: Primer synthesis, Escherichia coli DH5α, and BL21 (DE3) strains were purchased from Shanghai Biotechnology Co., Ltd., and the gene HorUox was synthesized by Qingke Biotechnology Co., Ltd. The pET28a (+) plasmid strain was preserved by the laboratory research group and is also a common commercially available plasmid.
[0048] (2) Main reagents: Endonucleases HindⅢ and EcoRⅠ were purchased from Takara; Hieff UniversalⅡOne Step Cloning Kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; potassium dihydrogen phosphate was purchased from Shanghai MacLean Biotechnology Co., Ltd.; anhydrous ethanol and chromatographic grade methanol were purchased from Tianjin Concord Technology Co., Ltd.; tryptone was purchased from Beijing Aoboxing Biotechnology Co., Ltd.; yeast extract powder was purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; agar, SDS, and Tris were purchased from BioFroxx, Germany; chromatographic grade uric acid was purchased from Sigma-Aldrich; the rest of the analytical reagents were purchased from Guangzhou Chemical Reagent Factory. DNA recovery kits and plasmid extraction kits were purchased from Guangzhou Meiji Biotechnology Co., Ltd.
[0049] (3) LB medium (Luria Broth Medium): 10.0 g of tryptone, 5.0 g of yeast extract powder, and 10.0 g of sodium chloride. Adjust the pH to 7.4 ± 0.2 with NaOH and dissolve in 1 L of ultrapure water. If solid culture medium is to be prepared, add 20 g of agar per liter of culture medium.
[0050] Implementation case 1: Construction of black yeast urate oxidase Escherichia coli engineering bacteria
[0051] 1. Synthesis of target gene
[0052] According to deep learning, a large number of urate oxidases were screened to obtain the black yeast (Hortaeawerneckii) urate oxidase gene sequence HorUox with high enzyme activity value. According to the codon preference of Escherichia coli and without changing its amino acid sequence, the urate oxidase gene sequence was optimized, and its sequence is shown in SEQ ID NO: 1. The amino acid sequence of black yeast urate oxidase HorUox is shown in SEQ ID NO: 2. The sequence of the 5-hydroxyisouridate hydrolase (Hydroxyisourate hydrolase) gene pucM from Cyberlindnera jadinii is shown in SEQ ID NO: 3, and the amino acid sequence encoded by it is shown in SEQ ID NO: 4. Primers with homology arms were designed according to the gene sequence (Table 1).
[0053] Table 1 Primer sequences
[0054]
[0055] 2. Construction of engineered Escherichia coli capable of degrading uric acid
[0056] Phanta Max Super-Fidelity DNA Polymerase (Vazyme) was used to perform PCR amplification reaction on the gene template, synthetic primers and other components according to the instructions to amplify the genes HorUox and pucM.
[0057] The amplified nucleotide sequences HorUox and pucM were connected with the linearized plasmid pET28a(+) treated with HindⅢ and EcoRⅠ using homologous recombinase to construct pET28a(+)-HorUm. Then, the cells were transformed into Escherichia coli DH5α competent cells by chemical heat shock. After the transformants grew out, single colonies were picked for colony PCR identification and sequencing confirmation. After sequence comparison, the sample sequencing results were correct, and its plasmid map was as follows Figure 1 .
[0058] The recombinant plasmid pET28a(+)-HorUm was heat-shocked into E. coli BL21(DE3) competent cells, and after the transformants grew out, single colonies were picked for colony PCR identification and sequencing confirmation. After sequence comparison, the sample sequencing results were correct. Therefore, the recombinant E. coli engineering bacteria BL21(DE3)-pET28a(+)-HorUm was successfully constructed.
[0059] Implementation case 2: Obtaining crude enzyme solution
[0060] Take out the recombinant E. coli engineering bacteria BL21(DE3)-pET28a(+)-HorUm from the -80℃ refrigerator and streak inoculate. After the colonies grow, pick a single colony and place it in a 5mL LB liquid test tube (containing 100μg / mL Kan r ) overnight. The next day, the seed solution was inoculated into 100 mL of LB liquid medium (containing 100 μg / mL Kan r ) and cultured in a shaker at 37°C and 200 rpm for 2-3 h until the OD of the bacterial solution reached 600 The temperature was 0.4-0.6, and the conical flask was placed in ice water for cooling. After the temperature dropped, IPTG was added to the culture medium at a final concentration of 0.5 mM, and the conical flask was transferred to a shaking incubator at 16°C and 150 rpm for induction for 24 hours.
[0061] After induction, the bacterial solution was placed in a 50 mL centrifuge tube and centrifuged at 4 ° C and 6000 rpm for 10 min using a low-temperature high-speed centrifuge. After centrifugation, the bacteria were collected and the supernatant was discarded. Resuspend and wash 3 times with 50 mM Tris-HCl buffer, and finally add 10 mL 50 mM Tris-HCl buffer to resuspend the bacteria. According to the proportion, add the super-splitting solution of hammer (ACE, Beijing, China) to react for a period of time. After obtaining the clarified fermentation solution, centrifuge at 4 ° C and 6000 rpm for 10 min, collect the supernatant, that is, the solution containing the target protein, that is, the crude enzyme solution, and finally store the solution in a 4 ° C refrigerator.
[0062] Implementation case 3: Determination of enzyme activity
[0063] The enzyme activity unit (U / L) is defined as: the amount of enzyme required to convert 1 μmol of uric acid per minute is one enzyme activity unit. 250 μL of enzyme solution and 500 μL of 2 mM uric acid solution were placed in a 1.5 mL centrifuge tube. The reaction temperature was 40°C and the reaction time was 10 min. After the reaction, 250 μL of stop solution (11% sulfuric acid solution) was added. The uric acid content after the enzyme reaction was detected by high performance liquid chromatography: the high performance liquid chromatograph was a Waters 2695 (Alliance system) liquid chromatograph, the chromatographic column was an Athena BST-C18 HPLC column (4.6×250 mm, 5 μm), the mobile phase ratio was 25 mM potassium dihydrogen phosphate buffer (adjusted to pH = 2.98 with phosphoric acid): methanol = 98:2, the flow rate was 1.0 mL / min, the detection wavelength was 284 nm, the injection volume was 10 μL, and the column temperature was 30°C. The uric acid standard curve is shown in the figure. Figure 2 The specific enzyme activity of the crude enzyme solution was measured to be 119.16±6.43U / g.
[0064] Implementation Case 4: Determination of the optimal reaction pH of enzymes
[0065] 100 μL crude enzyme solution, 250 μL 0.2 mM uric acid solution and 500 μL Tris-HCl buffer were placed in a 1.5 mL centrifuge tube. The reaction temperature was 40 ° C and the reaction time was 10 min. After the reaction, 150 μL of stop solution (11% sulfuric acid solution) was added. 200 μL of enzyme reaction solution was aspirated into a 96-well plate. The absorbance at 293 nm was measured using a continuous wavelength multifunctional microplate detection platform to calculate the enzyme activity. The highest point of enzyme activity was the optimal reaction pH. The measured maximum enzyme activity value was 100%. The relative enzyme activity at different pH values was calculated. Each sample was tested 3 times. The uric acid standard curve corresponding to the enzymatic method was established as follows: Figure 3 The buffers used were citric acid-sodium hydrogen phosphate buffer (pH 3.0-6.4), Tris-HCl buffer (pH 7.0-9.0), and glycine-sodium hydroxide buffer (pH 9.0-10.0). Figure 4 The enzyme activity is highest when the reaction pH is Tris-HCl buffer at pH 7.0. In addition, the enzyme can maintain a relatively high activity in a low acid and alkaline environment; in a strong acid environment, the relative activity of the enzyme is less than 40%.
[0066] Implementation Case 5: Determination of the Optimal Reaction Temperature of Enzymes
[0067] 1mL of total enzyme reaction system: 100μL of crude enzyme solution, 250μL of 0.2mM uric acid solution, 500μL of buffer solution, and 150μL of stop solution. Place the centrifuge tube at reaction temperatures of 30, 37, 40, 45, 50, 60, and 100℃ for 10min, add 150μL of stop solution (11% sulfuric acid solution) to terminate the reaction, mix evenly, and then pipette 200μL of enzyme reaction solution into a 96-well plate. Use a continuous wavelength multifunctional microplate detection platform to measure the absorbance at 293nm and calculate the activity of the enzyme. The highest point of enzyme activity is the optimal reaction temperature. The measured maximum enzyme activity value is 100%, and the relative enzyme activity at different temperatures is calculated, with the 100℃ high temperature enzyme as the control group. The results are as follows: Figure 5 , the reaction temperature is 40℃, the enzyme activity reaches the maximum value, so the optimal reaction temperature is 40℃.
[0068] Implementation Case 6: Construction of Mutant Engineering Bacteria
[0069] First, the server HotSpot Wizard was used to perform structural analysis, and all functional residues with high "variability" were marked as "hot spots". Then, the hot spots for the experiment were selected through molecular docking, molecular dynamics simulation, etc. Synthetic primers were designed based on the selected mutant amino acid hot spots.
[0070] Table 2 Mutation hotspot primers
[0071]
[0072] Plasmid ET28a(+)-HorUm was used as a template, and the primers in Table 2 were used for the amplification reaction. After the linear amplification fragment was completed, it was digested with DpnⅠ for half an hour. The system is shown in Table 3.
[0073] Table 3 DpnⅠ restriction enzyme system
[0074]
[0075] Use homologous recombinase to connect, transform the connected vector into E.coli DH5α competent cells, pick a single colony the next day for colony PCR identification and sequencing confirmation. After sequence comparison, the sample sequencing result is correct. The correctly sequenced transformants were inoculated, the plasmids were extracted, and transformed into Escherichia coli BL21 (DE3) cells to obtain engineered bacterial mutants BL21 (DE3)-pET28a (+) -HorUm (E183Y), BL21 (DE3) -pET28a (+) -HorUm (L287Y), and BL21 (DE3) -pET28a (+) -HorUm (D283R). Referring to the method of Implementation Case 3, the specific enzyme activities of the crude enzyme liquid of the mutant engineered bacteria were measured to be 241.06 ± 8.31 U / g, 206.44 ± 10.95 U / g, and 160.97 ± 20.79 U / g, respectively, which were 2.02 times, 1.73 times, and 1.35 times higher than those of the wild type, respectively.
[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A black yeast urate oxidase HorUox mutant, characterized in that: The amino acid sequence of the mutant is obtained by subjecting SEQ ID NO.2 to any of the following mutations: (1) E183Y; (2) L287Y; (3) D283R.
2. The black yeast urate oxidase HorUox mutant according to claim 1, characterized in that: The gene sequence encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.
1.
3. A gene encoding the HorUox mutant of black yeast urate oxidase according to claim 1 or 2.
4. The biomaterial related to the black yeast urate oxidase HorUox mutant according to any one of claims 1 to 2, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 3; (b) a recombinant expression vector containing the gene according to claim 3; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the gene according to claim 3; (e) a recombinant microorganism containing the expression cassette described in (a); (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).
5. The biomaterial according to claim 4, characterized in that: The starting vector of the recombinant expression vector in (b) and (c) is a pET series vector; The host microorganism corresponding to the recombinant microorganisms described in (d), (e) and (f) is selected from prokaryotes.
6. Use of the gene according to claim 3 or the biological material according to any one of claims 4 to 5 in preparing a mutant of urate oxidase HorUox from black yeast.
7. Use of the black yeast urate oxidase HorUox mutant according to any one of claims 1 to 2, the gene according to claim 3, or the biomaterial according to any one of claims 4 to 5 in the preparation of a product for degrading uric acid.
8. A method for obtaining the black yeast urate oxidase HorUox mutant according to claim 1 or 2, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on a gene encoding a black yeast urate oxidase HorUox having an amino acid sequence as shown in SEQ ID NO. 2 by designing primers containing mutation sites, and then expressing the gene to obtain the black yeast urate oxidase HorUox mutant according to claim 1 or 2.
9. The method according to claim 8, characterized in that: Primers containing mutation sites are designed to introduce mutations into the gene encoding the black yeast urate oxidase HorUox having an amino acid sequence as shown in SEQ ID NO.
2. After correct sequencing, the gene is transformed into Escherichia coli BL21 (DE3) for expression to obtain the black yeast urate oxidase HorUox mutant according to claim 1 or 2.