Use of dihydroorotase in reducing purine content

Abstract

The application belongs to the technical field of enzyme engineering, and discloses application of dihydroorotase in reducing purine content. Under specific reaction conditions, dihydroorotase can realize degradation of guanine. The reaction conditions include reaction pH, reaction temperature, and types and concentrations of catalysts. When it is applied to guanine degradation, the reaction pH is 7.0-10.0, the catalyst is a metal ion (Zn 2+ ), the concentration is 0.05-5 mM, and the reaction temperature is 20-45 DEG C. Catalytic reaction of purine substrates by the enzyme reaction conditions provided in the application can realize degradation of guanine, and the application lays a foundation for enzymatic preparation of low-purine food.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to the application of dihydroorotase in reducing purine content. Background Technology

[0002] Purines are a class of nitrogen-containing heterocyclic aromatic compounds, mainly including adenine, guanine, and two secondary metabolites, hypoxanthine and xanthine. They are ultimately metabolized into uric acid in the human body. Abnormal purine metabolism leads to an abnormal state where the blood uric acid level is higher than normal, known as hyperuricemia, which can subsequently cause gout. In recent years, the prevalence of hyperuricemia in my country has been rising continuously, with patients becoming younger, and it is usually closely related to various diseases, such as chronic kidney disease, periodontitis, heart failure, cardiovascular disease, hypertension, and diabetes.

[0003] High purine content in food is a cause of rapidly rising serum uric acid levels and gout attacks. Therefore, it is necessary to reduce the purine content in food. Methods for removing purines from food include adsorption, ultrasound, enzymatic addition, and microbial methods. Among these, enzymatic addition is not only specific and efficient, but its addition to flavoring mixtures can also remove purines during food preparation, showing broad application prospects.

[0004] However, there is currently limited research on purine-degrading enzymes, with the main focus being on reducing the purine content in food by reconstructing purine metabolic pathways in vitro.

[0005] Dihydroorotase (DHOase) belongs to the class of cyclic amide hydrolases. For example, the structure numbered 1J79 in the PDB database was obtained by X-ray diffraction analysis with a resolution of 1.70 Å, a molecular weight of 78.42 kDa, and a sequence length of 347. In the biosynthetic pathway, it catalyzes the reversible cyclization of N-carbamoyl-L-aspartic acid to L-dihydroorotate, which is the third step in pyrimidine biosynthesis. Under acidic conditions, this enzyme favors the dehydration of N-carbamoyl-L-aspartic acid to form L-dihydroorotate, while under alkaline conditions, it favors the hydrolysis and ring-opening of L-dihydroorotate to form N-carbamoyl-L-aspartic acid. Dihydroorotase exhibits significant structural and functional differences among different organisms (bacteria, fungi, higher plants, and mammals), possibly related to evolution and gene fusion. In higher eukaryotes, dihydroorotase is found in a large, multifunctional protein (CAD) that also contains carbamoyl phosphate synthase (CPSase) and aspartate transcarbamoylase (ATCase) catalyzing the first three reactions of the de novo pyrimidine pathway. In most prokaryotes, however, CPSase, ATCase, and DHOase are expressed and function independently; DHOase is a homodimer and monofunctional enzyme.

[0006] There are no reports of dihydroorotase playing a catalytic role in purine degradation. Summary of the Invention

[0007] To address the aforementioned shortcomings, one objective of this invention is to provide the application of dihydroorotic acidase in reducing purine content. The aim is to utilize dihydroorotic acidase to reduce purine content, thereby solving the problem of excessively high purine content in food and dietary restrictions for patients with hyperuricemia and gout.

[0008] In the aforementioned application, the purine may specifically be the purine found in food;

[0009] The purine in question may specifically be guanine.

[0010] Another object of the present invention is to provide a method for reducing the purine content in food.

[0011] The method for reducing the purine content in food provided by this invention includes the following steps:

[0012] By using genetic engineering to express dihydroorotase, the enzymatic reaction conditions can be altered, thereby reducing the purine content in food.

[0013] In the above method, the purine may specifically be guanine;

[0014] The enzyme-catalyzed reaction conditions include catalyst type, catalyst concentration, reaction pH, and reaction temperature;

[0015] Furthermore, the catalyst type includes Zn. 2+ Na + K + Mg 2+ Fe 3+ Ca 2+ Cu 2+ At least one of them;

[0016] Furthermore, the catalyst concentration is 0.05-5 mM, specifically 0.05, 0.5, 5, 10, or 50 mM;

[0017] Furthermore, the pH of the reaction is 7.0-10.0;

[0018] Furthermore, the reaction temperature is 20-45℃;

[0019] The activity of the dihydroorotase was 789.8 U / mL.

[0020] The above method is operated as follows: at the reaction temperature, guanine or food containing guanine is placed in an alkaline reaction system containing dihydroorotase and a catalyst to carry out the reaction.

[0021] The catalyst is Zn. 2+ Na + K + Mg 2+ Fe 3+ Ca 2+ Cu 2+ At least one of the following: the concentration of the catalyst is 0.05-5 mM, specifically 0.05-2 mM; the pH of the alkaline reaction system is 7.0-10.0, specifically 7.5-8.5; the reaction temperature is 20-45℃, specifically 20-30℃, more specifically 25℃.

[0022] In the alkaline reaction system, the activity of dihydroorotase is 50-200 U / mL, and the concentration of guanine is 5-200 mg / L.

[0023] The application of dihydroorotase and the above-mentioned methods for reducing purine content in the preparation of low-purine foods is also within the scope of protection of this invention.

[0024] The purine in question may specifically be guanine.

[0025] The present invention has the following advantages:

[0026] This invention discovers that dihydroorotase has a novel function. In addition to playing a role in the pyrimidine biosynthesis pathway, it can reduce the content of guanine under specific reaction conditions. When applied to the food processing field, it can be used to prepare low-purine nutritional health foods, supplementing the enzymatic removal of purines from food, thereby achieving personalized dietary control for hyperuricemia and gout.

[0027] This invention studies the activity of dihydroorotase in degrading purines, which can supplement the enzymatic reduction of purines in food, solve the problem of kinetic incompatibility between different component enzymes in multi-enzyme complexes, and lay the foundation for the development of low-purine foods. Attached Figure Description

[0028] Figure 1 This is the gene map of the dihydroorotase recombinant expression vector in Example 1 of the present invention.

[0029] Figure 2The SDS-PAGE results for the induced expression and purification of recombinant dihydroorotase in Examples 1 and 2 of this invention are shown. Lane M is a 130kD marker, lane 1 is the crude enzyme solution, and lanes 2-16 are AKTA purification elution buffers (imidazolium concentrations of 20mM, 20mM, 50mM, 50mM, 100mM, 100mM, 200mM, 200mM, 200mM, 200mM, 300mM, 300mM, 400mM, 400mM, 500mM, and 500mM, respectively).

[0030] Figure 3 Different Zn in Embodiment 3 of the present invention 2+ The effect of concentration on the degradation of purines by dihydroorotase, where a, b, c, and d represent adenine, guanine, hypoxanthine, and xanthine, respectively.

[0031] Figure 4 This illustrates the effect of pH on dihydroorotase activity in Example 4 of the present invention.

[0032] Figure 5 This illustrates the effect of temperature on dihydroorotase activity in Example 5 of the present invention.

[0033] Figure 6 This is for the determination of the pH stability of dihydroorotase in Example 6 of the present invention.

[0034] Figure 7 This is for the determination of the temperature stability of dihydroorotase in Example 7 of the present invention.

[0035] Figure 8 This is a determination of the degradation rate of guanine by dihydroorotase under optimal reaction conditions in Example 8 of the present invention.

[0036] Figure 9 This invention relates to Example 9, which describes the determination of the degradation rate of guanine by dihydroorotase in soy milk. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] In the examples, purines were used as substrates to determine the activity of dihydroorotase.

[0040] The amount of enzyme required to degrade 1 mg of guanine within 24 hours at a temperature of 25°C and a pH of 8 is defined as one enzyme activity unit (U).

[0041] The purine determination method in the examples is as follows:

[0042] The analysis was performed using an Agilent 1260 high-performance liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) and an InertSustain C18 column (5 μm × 250 mm × 4.6 mm). The injection volume was 20 μL, the flow rate was 0.8 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. Mobile phase A was ultrapure water, and mobile phase B was ultrapure water:methanol:glacial acetic acid:25% tetrabutylammonium hydroxide (882:100:15:3, v / v), with a mobile phase A:mobile phase B ratio of 50:50.

[0043] Example 1: Induced expression of dihydroorotase

[0044] The dihydroorotase gene sequence was obtained from the RCSB PDB (https: / / www.rcsb.org / ) database, with PDB ID 1J79. The obtained dihydroorotase gene was inserted into the expression vector pET-28a, with the restriction enzyme site BamHI-Xhol, and a His tag was added to facilitate subsequent protein purification. After sequencing verification, the recombinant expression vector pET-28a / dihydroorotase was obtained. The gene map is shown below. Figure 1 As shown, Feature 16 is the target gene. After the recombinant expression vector was synthesized and verified to be correct by colony PCR and sequencing, it was transformed into Escherichia coli BL21(DE3) using the heat shock transformation method. The transformed recombinant E. coli BL21(DE3) was stored in an ultra-low temperature freezer at -80°C.

[0045] Subsequently, the recombinant Escherichia coli was inoculated into LB liquid medium containing kanamycin sulfate (working concentration 50 ng / μL) and cultured at 220 rpm and 37°C until OD500. 600nm=0.4-0.6. IPTG was added to the bacterial culture to a final concentration of 1 mM. After incubation at 16℃ for 20 h, the bacterial cells were collected by centrifugation and resuspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 8.0). The cells were then sonicated on ice under the following conditions: power 210 W, sonication for 3 seconds, interval 3 seconds, for 30 min, until the bacterial culture changed from a viscous emulsion to a clear state. After sonication, the culture was centrifuged at 12000 rpm for 30 min, and the supernatant was collected as the crude enzyme solution. Enzyme expression was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows: Figure 2 As shown.

[0046] The results showed that the target protein band was present in the supernatant (crude enzyme solution) after cell lysis, indicating that the protein was successfully expressed and mainly existed in the form of soluble protein.

[0047] Example 2: Purification of dihydroorotase

[0048] To characterize the properties of dihydroorotase, a nickel ion affinity column, HisTrap, was used. TM Purification of dihydroorotase using an HP column (1cm×1cm) (GE Healthcare, MA, USA).

[0049] The purification efficiency of the enzyme was analyzed using SDS-PAGE, such as... Figure 2 As shown, the theoretical molecular weight of the protein is 39.21 kDa, while the actual molecular weight is 41 kDa. This is likely because the recombinant protein has 6×His tags added to both the N-terminus and C-terminus to facilitate protein purification. At this point, purified dihydroorotase has been obtained.

[0050] Example 3, Zn 2+ Effect of concentration on dihydroorotase activity

[0051] In order to study Zn 2+ The effect of concentration on dihydroorotase activity was investigated by adding ZnCl2 to the reaction system. 2+ The concentrations were 0, 0.5, 1, 2, and 3 mM, with a total reaction volume of 250 μL. 50 μL of enzyme solution (789.8 U / mL) was mixed with 50 μL of purine solution (800 mg / L), and ZnCl2 solution (25 mM) was added to make Zn... 2+The concentrations were 0, 0.5, 1, 2, and 3 mM, and the volume was brought up to 250 μL with Tris-HCl buffer (100 mM, pH 8.0). The reaction was carried out at 25 °C for 24 h, and then the reaction solution was heated at 100 °C for 10 min to terminate the reaction. The activity of dihydroorotase was determined by measuring the degradation rate of purine substrates.

[0052] The results are as follows Figure 3 As shown, Zn is added to the reaction system. 2+ Activate the catalytic reaction, Zn 2+ At concentrations that are too high or too low, this dihydroorotase has no significant degradation effect on any of the four purines, while when Zn... 2+ At a concentration of 0.5 mM, the recombinant enzyme can degrade about 20% of guanine after 24 hours, at which point the degradation effect is the best. However, it has no obvious degradation effect on the other three purines. Therefore, the subsequent determination of enzymatic properties is only for guanine.

[0053] Example 4: Effect of pH on the activity of dihydroorotase

[0054] To investigate the effect of pH on the activity of dihydroorotase, the reaction was carried out in Tris-HCl buffer (100 mM) with a pH range of 7.0–9.0 and glycine-NaOH buffer (50 mM) with a pH range of 9.0–10.0, with a total reaction volume of 250 μL. 50 μL of enzyme solution (789.8 U / mL) was mixed with 50 μL of purine solution (800 mg / L), and 5 μL of ZnCl2 solution (25 mM) was added. The volume was then adjusted to 250 μL using either Tris-HCl buffer or glycine-NaOH buffer, and the reaction was carried out at 25 °C for 24 h. The reaction was then terminated by heating the solution at 100 °C for 10 min, and the activity of dihydroorotase was determined by measuring the degradation rate of the purine substrate.

[0055] result( Figure 4 The results showed that within the pH range of 7.0-10.0, the activity of this dihydroorotase first increased and then decreased with increasing pH, with the highest enzyme activity at pH 8.0, at which point it could degrade approximately 46% of guanine. Therefore, the optimal reaction pH for the enzyme is 8.0. When the pH is less than 7.5 or higher than 8.5, the enzyme activity rapidly decreased, and there was virtually no activity at pH 9.0-10.0, indicating that this enzyme is more suitable for use in a weakly alkaline environment.

[0056] Example 5: Effect of temperature on dihydroorotase activity

[0057] To investigate the effect of temperature on dihydroorotase activity, the reaction was carried out in Tris-HCl buffer (pH 8.0, 100 mM) in a total volume of 250 μL. 50 μL of enzyme solution (789.8 U / mL) was mixed with 50 μL of purine solution (800 mg / L), and 5 μL of ZnCl2 solution (25 mM) was added. The volume was then brought to 250 μL using Tris-HCl buffer. The temperature range was set at 20–45 °C, and the reaction was allowed to proceed for 24 h. The reaction was then terminated by heating the solution at 100 °C for 10 min. The activity of dihydroorotase was determined by measuring the degradation rate of the purine substrate.

[0058] result( Figure 5 The results showed that, within the temperature range of 20-45℃, the activity of this dihydroorotase initially increased and then decreased with increasing temperature. The enzyme activity was highest at a reaction temperature of 25℃, thus determining the optimal reaction temperature to be 25℃. Within the temperature range of 20-30℃, more than 60% of the maximum enzyme activity was retained; however, the enzyme activity began to decrease sharply above 30℃.

[0059] Example 6: Determination of pH stability of dihydroorotase

[0060] To investigate the pH stability of this dihydroorotase, the enzyme solution (789.8 U / mL) was placed in buffer solutions with different pH values ​​(7, 7.5, 8, 8.5, 9, 9.5, 10) and incubated at 4°C for 24 h. Residual activity was then measured. After incubation, 50 μL of the enzyme solution was mixed with 50 μL of purine solution (800 mg / L), and 5 μL of ZnCl2 solution (25 mM) was added. The volume was then brought to 250 μL using Tris-HCl buffer (pH 8.0, 100 mM), and the reaction was carried out at 25°C for 24 h. The reaction solution was then heated at 100°C for 10 min to terminate the reaction. The activity of the dihydroorotase was determined by measuring the degradation rate of the purine substrate.

[0061] result( Figure 6 The results showed that after incubation at pH 7.0-9.0 for 24 hours, the relative enzyme activity of this dihydroorotase remained above 40%, and at pH 8.5, the relative enzyme activity was maintained at 90%. However, the enzyme activity decreased rapidly at pH above 9.0. These results indicate that heterologously expressed dihydroorotase is relatively stable in a weakly alkaline environment.

[0062] Example 7: Determination of the temperature stability of dihydroorotase

[0063] To investigate the temperature stability of this dihydroorotase, the enzyme solution (789.8 U / mL) was incubated at 4, 20, 30, and 40 °C for 24 h, and its residual activity was measured. After incubation, 50 μL of the enzyme solution was mixed with 50 μL of purine solution (800 mg / L), and 5 μL of ZnCl2 solution (25 mM) was added. The volume was then brought to 250 μL using Tris-HCl buffer (pH 8.0, 100 mM), and the reaction was carried out at 25 °C for 24 h. The reaction solution was then heated at 100 °C for 10 min to terminate the reaction, and the degradation rate of the purine substrate was measured to determine the activity of the dihydroorotase.

[0064] result( Figure 7 The results showed that the lower the temperature, the more stable the enzyme. After incubation at 4-20℃ for 24 hours, the enzyme retained more than 60% of its activity. However, after incubation at 40℃ for 24 hours, the enzyme activity essentially decreased to zero. Excessively high temperatures may alter the protein's conformation, causing it to lose its activity. These results indicate that the enzyme is not heat-resistant and is more suitable for use in medium- and low-temperature environments.

[0065] Example 8: Determination of Guanine Degradation Rate by Dihydroorotase under Optimal Reaction Conditions

[0066] Mix 50 μL of dihydroorotase enzyme solution (789.8 U / mL) with 50 μL of purine solution (800 mg / L), add 5 μL of ZnCl2 solution (25 mM), and bring the volume to 250 μL using Tris-HCl buffer (pH 8.0, 100 mM). Incubate the mixture at 25 °C for 24 h. Then, heat the reaction solution at 100 °C for 10 min to terminate the reaction. Finally, determine the activity of dihydroorotase by measuring the degradation rate of the purine substrate.

[0067] result( Figure 8 The results showed that, under optimal reaction conditions, the dihydroorotase could degrade guanine by 46%.

[0068] Example 9: Determination of the degradation rate of guanine in soy milk by dihydroorotase

[0069] Take 1950 μL of soy milk, add 50 μL of ZnCl2 solution (25 mM) and 500 μL of dihydroorotase (789.8 U / mL). React at 25℃ for 24 h. After the reaction is complete, extract the purines from the soy milk, and then determine the purine degradation rate to determine the activity of dihydroorotase. The specific steps for purine extraction from soy milk are as follows: Take 500 μL of the reacted sample, add 2.5 mL each of trifluoroacetic acid and formic acid, incubate at 85℃ for 15 min, then rapidly cool in an ice-water bath, transfer to a rotary evaporator flask, and rotary evaporate to near dryness at 60℃. Redissolve the residue with the mobile phase and bring the volume to 5 mL. Centrifuge at 9000 r / min for 10 min, collect the supernatant, filter through a 0.22 μm organic microporous membrane, and then perform high-performance liquid chromatography (HPLC) detection.

[0070] result( Figure 9 The results showed that the dihydroorotase had a 14% degradation rate of guanine in soy milk.

[0071] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of dihydroorotase in reducing purine content; In this application, the purine is the purine found in food; The purine in question is guanine; The enzymatic reaction conditions in the application include catalyst type, catalyst concentration, reaction pH, and reaction temperature; The catalyst is Zn. 2+ ; The catalyst concentration is 0.05-5 mM; The reaction is carried out at a pH of 7.0-10.0; The reaction temperature is 20-45℃.

2. A method for reducing the guanine content in food, comprising the following steps: By using genetic engineering to express dihydroorotase, the enzymatic reaction conditions can be altered, thereby reducing the purine content in food. The enzyme-catalyzed reaction conditions include catalyst type, catalyst concentration, reaction pH, and reaction temperature; The catalyst is Zn. 2+ ; The catalyst concentration is 0.05-5 mM; The reaction is carried out at a pH of 7.0-10.0; The reaction temperature is 20-45℃.

3. The method according to claim 2, characterized in that, The activity of the dihydroorotase was 789.8 U / mL.

4. The method according to claim 2, characterized in that, The method is operated as follows: at the reaction temperature, guanine or food containing guanine is placed in an alkaline reaction system containing dihydroorotase and a catalyst to carry out the reaction.

5. The method according to claim 4, characterized in that, In the alkaline reaction system, the activity of dihydroorotase is 50-200 U / mL, and the concentration of guanine is 5-200 mg / L.

6. The use of dihydroorotase or the method for reducing the guanine content in food according to any one of claims 2-5 in the preparation of low-purine foods.

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