Immobilized nucleosidase mutant and application thereof

Abstract

The invention discloses an immobilized nucleoside enzyme mutant and application thereof. The immobilized nucleoside enzyme mutant is prepared by covalently immobilizing a nucleoside enzyme mutant G178D / A202K on aminated magnetic Fe3O4 nanoparticles. The catalyst is applied to a hydrolysis reaction of guanine nucleotide (GMP), efficient catalysis is carried out in a water phase at 60-70 DEG C, the catalyst can be rapidly separated and recovered through an external magnetic field after the reaction, and then high-purity guanine is obtained through solid-liquid separation. The immobilized enzyme can be repeatedly used for more than or equal to 8 times, still keeps more than or equal to 80% of activity, remarkably improves the industrial efficiency of guanine preparation, and has the advantages of green process, simplicity and convenience in operation, low cost, easiness in industrialization and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis, specifically relating to an immobilized nucleoside enzyme mutant and its applications. Background Technology

[0002] Guanine is an important purine pharmaceutical intermediate, especially in the synthesis of antiviral drugs (such as acyclovir and ganciclovir), where it holds irreplaceable core value. Currently, the main methods for producing guanine include chemical synthesis, natural raw material extraction, and the increasingly popular bio-enzymatic catalysis method.

[0003] Traditional chemical synthesis methods generally suffer from harsh reaction conditions, long process routes, and severe environmental pollution. For example, the preparation of guanine from guanidine nitrate and ethyl cyanoacetate via a multi-step cyclization reaction (CN100491377), or the ring-closing reaction of 2,4,5-triamino-6-hydroxypyrimidine sulfate as an intermediate with formic acid, formamide, and other reagents at high temperatures (CN106117207), both involve high temperature and high pressure, the use of organic solvents, and numerous side reactions, leading to difficulties in product purification, unstable yields, and significant environmental impact. While natural extraction methods (such as extraction from fish scales and other natural raw materials) have a wide range of raw material sources, they suffer from limitations such as complex extraction processes, low yields, and difficulty in controlling product purity, making it difficult to meet the needs of large-scale industrial production.

[0004] In recent years, bio-enzymatic catalysis has become a research hotspot for the green synthesis of guanine due to its advantages such as mild reaction conditions, high specificity, and environmental friendliness. Existing technologies have already developed methods for preparing guanine by enzymatically hydrolyzing purine nucleosides (such as guanosine). For example, CN115011646A discloses a process for preparing guanine and D-ribose by hydrolyzing guanosine using guanosine hydrolase; CN116515921A further employs a combined enzymatic hydrolysis of guanosine using guanosine hydrolase and neutral protease. These methods demonstrate the advantages of enzymatic synthesis to some extent. However, in industrial applications, free enzyme catalysis still faces significant challenges: (1) enzymes are easily deactivated in the reaction system, especially in complex systems with high temperatures or solid products, resulting in poor stability; (2) after the reaction, free enzymes are difficult to efficiently separate and recover from the reaction system, leading to low enzyme utilization and high production costs; (3) when the product is a solid with extremely poor water solubility (such as guanine, which has extremely low solubility in water), the separation of free enzymes from solid products is particularly difficult, increasing the complexity and cost of subsequent separation and purification processes. Furthermore, there are still no reports on efficient and stable enzyme catalytic processes for the direct hydrolysis of guanine nucleotides (such as GMP) to prepare guanine. In particular, immobilizing thermostable enzymes suitable for this reaction to achieve convenient recovery and reuse of the catalyst is crucial for industrial production.

[0005] Immobilized enzyme technology is an effective strategy for improving enzyme stability and enabling enzyme reuse. Among them, magnetic Fe3O4 nanoparticles, as an emerging immobilization carrier, show great potential in the field of enzyme immobilization due to their superparamagnetism, high specific surface area, ease of surface functionalization, and good biocompatibility. The immobilized enzyme can be rapidly and completely separated from the reaction system using an external magnetic field, a characteristic particularly suitable for catalytic processes where the product is a solid or the system has high viscosity. Summary of the Invention

[0006] Based on this, the inventors first obtained a heat-resistant nucleoside enzyme mutant G178D / A202K through rational design. This mutant can efficiently catalyze the hydrolysis of guanine nucleotides (GMP) to guanine at the optimal temperature of 70℃ (see reaction mechanism). Figure 1 This high-temperature resistant nucleoside enzyme mutant exhibits excellent thermal stability. By immobilizing this mutant on a magnetic Fe3O4 nanocarrier, a novel biocatalytic method for the preparation of guanine was constructed, possessing high catalytic activity, excellent operational stability, and reusability, enabling the green, continuous, and low-cost industrial biomanufacturing of guanine.

[0007] The technical solution of this invention:

[0008] A thermostable nucleoside enzyme mutant, G178D / A202K, has the amino acid sequence shown in SEQ ID NO: 2.

[0009] A nucleic acid molecule encoding the above-mentioned thermostable nucleoside enzyme mutant G178D / A202K.

[0010] An expression vector containing the aforementioned nucleic acid molecules.

[0011] A recombinant cell comprising the above-described expression vector.

[0012] An immobilized nucleoside enzyme mutant is constructed by covalently immobilizing a heat-resistant nucleoside enzyme mutant on a surface-modified magnetic Fe3O4 nanoparticle carrier; the heat-resistant nucleoside enzyme mutant is the nucleoside enzyme mutant G178D / A202K with the amino acid sequence shown in SEQ ID NO: 2.

[0013] Preferably, the magnetic Fe3O4 nanoparticle carrier is Fe3O4 nanoparticles modified with an aminosilane reagent and rich in amino functional groups on the surface.

[0014] Preferably, the aminosilane reagent is 3-aminopropyltriethoxysilane.

[0015] This invention also provides a method for preparing an immobilized nuclease mutant, comprising the following steps:

[0016] S1. Preparation of magnetic Fe3O4 nanoparticles;

[0017] S2. The Fe3O4 nanoparticles obtained in step S1 were modified by surface amylation using an aminosilane reagent to obtain an NH2-Fe3O4 support.

[0018] S3. Disperse the NH2-Fe3O4 support in a buffer solution, add the cross-linking agent glutaraldehyde for activation, and obtain an aldehyde-activated magnetic support;

[0019] S4. Mix the enzyme solution containing the nuclease mutant G178D / A202K with the activated vector obtained in step S3, and incubate to covalently immobilize the enzyme on the vector.

[0020] S5. After incubation, the immobilized nucleoside enzyme mutant is obtained by separation and washing using a magnetic field.

[0021] Preferably, in step S1, the magnetic Fe3O4 nanoparticles are prepared by a co-precipitation method;

[0022] In step S4, the incubation conditions are as follows: shake in a buffer solution at 4-25℃ and pH 7.0-8.5 for 4-16 hours.

[0023] The preparation method of the enzyme solution containing the nuclease mutant G178D / A202K is as follows: A recombinant genetically engineered bacterium containing the nuclease mutant gene is constructed, inoculated into a fermentation medium after seed culture, and fermented. After fermentation, the bacterial cells are collected by centrifugation. The cells are resuspended in buffer solution, sonicated, and centrifuged again. The supernatant is the crude enzyme solution of the nuclease mutant G178D / A202K.

[0024] Application of the immobilized nucleoside enzyme mutant and the thermostable nucleoside enzyme mutant G178D / A202K in catalyzing the hydrolysis of guanine nucleotides to guanine.

[0025] A method for preparing guanine using an immobilized nuclease mutant includes the following steps:

[0026] (1) Using water or buffer solution as the reaction medium, add the substrate guanine nucleotide and the immobilized nuclease mutant described above;

[0027] (2) The reaction system of step (1) is subjected to hydrolysis at 50-70℃ with stirring until the substrate is completely converted;

[0028] (3) After the reaction is completed, the immobilized nuclease mutant is separated from the reaction system and recovered using an external magnetic field;

[0029] (4) The reaction liquid after separating the catalyst in step (3) is subjected to solid-liquid separation, the solid part is collected, washed and dried to obtain guanine product.

[0030] Preferably, in step (1), the concentration of the substrate guanine nucleotide is 100-250 g / L, and the amount of the immobilized nuclease mutant added is 10-25 g / L based on the dry weight of the vector;

[0031] Preferably, in step (2), the temperature of the catalytic reaction is 60-70°C and the reaction time is 2-5 hours;

[0032] Preferably, the immobilized nuclease mutant recovered in step (3) can be reused in a new round of reaction in step (1) after washing, and can be reused at least 8 times while still maintaining ≥80% activity.

[0033] Beneficial effects:

[0034] This invention utilizes magnetic Fe3O4 nanoparticles to immobilize a high-temperature resistant nucleoside enzyme mutant, significantly improving the enzyme's thermal and operational stability. It also enables rapid magnetic separation in solid systems where guanine is abundant, avoiding the problem of free enzyme being adsorbed and inactivated by the product. The immobilized enzyme maintains high catalytic efficiency even under high substrate concentrations (100-250 g / L), achieving a GMP conversion rate of over 99%, and can be reused more than 8 times while retaining over 80% of its activity. This method is simple to operate, environmentally friendly, and significantly reduces enzyme costs, providing a highly efficient and economical new approach for the industrial production of guanine. Attached Figure Description

[0035] Figure 1 The reaction mechanism for preparing guanine by nucleoside enzyme hydrolysis of GMP;

[0036] Figure 2 The effect of temperature on the enzyme activity of the nucleoside enzyme mutant G178D / A202K;

[0037] Figure 3 Thermostability of the nucleoside enzyme mutant G178D / A202K;

[0038] Figure 4 This is an SDS-PAGE image of protein expression of the nuclease mutant G178D / A202K in Escherichia coli BL21(DE3). Lane descriptions: M is the standard marker; Lane 1 is the supernatant after heat treatment at 50℃; Lane 2 is the supernatant after heat treatment at 60℃; Lane 3 is the supernatant after heat treatment at 70℃.

[0039] Figure 5The liquid chromatography results of the immobilized nuclease mutant G178D / A202K catalyzing the hydrolysis of GMP to guanine are shown in Figure a. Figure a shows the liquid chromatography peaks at 0 h, and Figure b shows the liquid chromatography peaks at 5 h. Detailed Implementation

[0040] To better illustrate the purpose, technical solution, and advantages of this invention, the following embodiments are provided for further detailed description. It should be understood that the specific embodiments described herein are merely illustrative of the invention, and the scope of protection of this invention is not limited thereto.

[0041] Example 1: Preparation of nucleoside enzyme free enzyme

[0042] By performing three-dimensional structural simulation and substrate docking analysis on the amino acid sequence of the nuclease (PpnN, NCBI accession number MBT4838044.1) derived from *Methylococcales bacterium* (as shown in SEQ ID NO.1), a site-directed mutagenesis was performed at key sites selected at the substrate binding site using a rational design method. Glycine (G) at position 178 was mutated to aspartic acid (D), and alanine (A) at position 202 was mutated to lysine (K), resulting in the thermostable nuclease mutant G178D / A202K, whose amino acid sequence is shown in SEQ ID NO.2. The recombinant genetically engineered bacterium *E. coli* BL21-pET28a-G178D / A202K containing the nuclease mutant gene (codon optimized) was directly synthesized by General Biotechnology (Anhui) Co., Ltd., and stored at -80°C.

[0043] The recombinant *E. coli* strain described above was inoculated into 10 ml of LB medium containing kanamycin (50 μg / mL) resistance and cultured at 37°C for 12 h. Then, it was transferred to 400 mL of TB fermentation medium at a 2% inoculation rate. The medium was first incubated at 37°C and 200 rpm until the bacterial OD600 reached 0.6, then transferred to 30°C and 200 rpm for shake-flask induction fermentation for 18 h. After fermentation, the bacterial cells were collected by centrifugation at 8000 rpm for 5 min. 4 g of the fermented wet bacterial cells were weighed, resuspended in 40 ml of 50 mM Thris pH 8.0 buffer, and sonicated (1 s, 2 s, 20 min). The mixture was then centrifuged at 8000 rpm for 20 min, and the supernatant was the crude enzyme solution of the nucleoside enzyme mutant G178D / A202K.

[0044] Example 2: Enzyme purification

[0045] To purify the wild-type nucleoside enzyme PpnN and its mutant G178D / A202K, an AKTAprime chromatography system from GE was used, employing a 5 mL HisTrap HP nickel column for affinity chromatography. The column was pre-equilibrated with pH 8.5, 0.5 M NaCl, 20 mM imidazole, and 20 mM sodium phosphate buffer (buffer A). The elution buffer was pH 8.5, 0.5 M NaCl, 0.5 M imidazole, and 20 mM phosphate buffer (buffer B), using a gradient elution from 0% to 100% buffer B for a total elution time of 60 min. The collected active protein was then further desalted using a Sephacryl S-300 gel column to remove contaminating proteins. The target protein was collected at high concentrations using UV detection. The purified enzyme was stored at -80°C for subsequent determination of protein concentration and activity.

[0046] Example 3: Enzyme activity assay of wild-type PpnN and mutant enzymes

[0047] Take two 50ml shake flasks, weigh 2g of GMP sodium salt (final concentration 100g / L) into each, add 19ml of water and stir well. Add 1ml of the purified enzyme solution obtained in Example 2 above to each flask, and immediately transfer to a 250rpm, 60℃ constant temperature water bath shaker for reaction. After 20min, take 1ml of each sample and heat-treat in boiling water for 10min, centrifuge at 12000rpm for 5min, take the supernatant for liquid chromatography analysis, calculate the amount of GMP converted, and obtain the specific enzyme activity of the two enzymes.

[0048] Enzyme activity is defined as the amount of enzyme that converts 1 μmol of GMP substrate per minute at 60°C, defined as one unit of enzyme activity (U). The relative enzyme activity of mutants is compared with the wild-type enzyme activity as 100%.

[0049] HPLC determination of guanine nucleotides: Chromatographic conditions: Column: Cosmosil PAQ C18 (250 X 4.6 mm, 5 μm), flow rate: 1 mL / min, wavelength: 254 nm, column temperature: 30 °C, injection volume: 20 μL, mobile phase: (0.1 M phosphate buffer, pH 6.0): methanol = 96:4

[0050] The results showed that the enzyme activity of the mutant G178D / A202K was 2.1 times higher than that of the wild-type PpnN.

[0051] Example 4: Effect of temperature on enzyme activity of mutant G178D / A202K

[0052] 4.1 Determining the optimal reaction temperature of the enzyme

[0053] Six reaction systems identical to those in Example 3 were set up, with the only difference being that the water bath shaking temperature was sequentially changed to 50℃, 60℃, 65℃, 70℃, 75℃, and 80℃. The reactions were stopped after 30 minutes, and samples were taken for analysis to calculate enzyme activity. The highest enzyme activity was taken as 100%, and relative enzyme activities were compared to plot a temperature-relative activity curve. The sample with the highest enzyme activity was used as the relative enzyme activity 100%, and relative enzyme activities were compared. Figure 2 It is known that the optimal reaction temperature for the mutant G178D / A202K enzyme is 70℃.

[0054] 4.2 Determination of enzyme thermostability

[0055] To investigate the thermostability of the enzyme, the purified mutant G178D / A202K enzyme obtained according to the purification method described in Example 2 was incubated in water baths at 50°C, 60°C, and 70°C, respectively. Residual enzyme activity was measured every 1 hour using the enzyme activity detection method described in Example 3. The initial detected enzyme activity was taken as 100% relative enzyme activity, and the enzyme activity of subsequent samples was compared.

[0056] Results (e.g.) Figure 3 The mutant G178D / A202K exhibits excellent thermal stability. No decrease in activity was detected after incubation at 50℃ for 2 hours, and the residual activity remained above 90% after incubation at 60℃ for 2 hours. After incubation at 70℃ for 2 hours, the activity remained above 80%.

[0057] The crude enzyme of mutant G178D / A202K was incubated at 50℃-70℃ for 20 min, centrifuged, and the supernatant was subjected to SDS-PAGE electrophoresis. The results are as follows. Figure 4 As shown in lanes 1-3, a complete and distinct specific band remains at 52 kDa, further demonstrating that the mutant G178D / A202K exhibits good thermal stability at 70°C. Therefore, its thermal stability can be utilized to heat-treat the crude enzyme solution obtained in Example 3 at 70°C for 20 minutes. After simple centrifugation, a high-purity enzyme solution can be obtained for subsequent immobilization enzyme operations.

[0058] Example 5: Preparation of Aminated Magnetic Fe3O4 Nanoparticles (NH2-Fe3O4)

[0059] A chemical coprecipitation method was employed: FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water in a specific molar ratio, and the mixture was stirred vigorously under nitrogen protection and heated to 80°C. Excess ammonia was rapidly added, and the reaction was allowed to proceed for 1 hour. The nanoparticles were then magnetically separated and washed with deionized water until neutral to obtain Fe3O4 nanoparticles. The obtained Fe3O4 nanoparticles were redispersed in an ethanol-water mixed solvent, and 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 60°C for 6 hours. After the reaction, the nanoparticles were magnetically separated, washed several times with ethanol, and dried under vacuum to obtain the NH2-Fe3O4 support.

[0060] Example 6: Preparation of immobilized nucleoside enzyme mutant G178D / A202K

[0061] Weigh 100 mg of NH₂-Fe₃O₄ prepared in Example 7 and disperse it in 10 mL of phosphate-buffered saline (PBS) at pH 8.0. Add 1 mL of 5% (v / v) glutaraldehyde solution and activate by shaking at room temperature for 2 hours. Magnetic separation is performed, and the mixture is washed three times with PBS buffer to remove unreacted glutaraldehyde. The activated vector is redispersed in 10 mL of PBS, and 10 mL of PBS solution containing 50 mg of the heat-treated, simply purified nucleoside enzyme mutant G178D / A202K is added. The mixture is then incubated with slow shaking at 4°C for 12 hours. After incubation, the immobilized enzyme is separated by magnetic adsorption and washed thoroughly with PBS buffer until no protein is detected in the washings, yielding the immobilized nucleoside enzyme mutant, which is stored at 4°C for later use.

[0062] Example 7: Preparation of Guanine using Immobilized Nucleotidase Mutants

[0063] Add 90 mL of deionized water to a 250 mL Erlenmeyer flask, and add 25 g of GMP sodium salt while stirring until completely dissolved and suspended. Add the immobilized nuclease mutant (2.5 g based on the dry weight of the vector) prepared according to method 6. Place the reaction system in a constant temperature shaker at 65 °C and 200 rpm. Take samples at regular intervals, inactivate the GMP in a boiling water bath, centrifuge, and collect the supernatant. Analyze the GMP residue using HPLC. After 5 hours of reaction, take samples for liquid chromatography analysis. The results are as follows: Figure 5 As shown, the GMP conversion rate reached 99.5%. After the reaction, the conical flask was placed next to a strong magnet, and the immobilized enzyme was rapidly adsorbed onto the flask wall. The upper part of the reaction solution containing guanine solid suspension was poured off. The reaction solution was filtered, and the filter cake was washed twice with hot water and dried under vacuum at 60°C for 12 hours to obtain 21.5 g of white guanine powder. The purity was greater than 99.5% and the molar yield was approximately 97%, as determined by HPLC.

[0064] Example 8: Reusability of Immobilized Nuclease Mutants

[0065] The immobilized enzyme recovered by magnetic field in Example 7 was washed three times with deionized water and then directly used in a new round of reaction. The reaction conditions were the same as in Example 7. The above reaction-magnetic recovery-washing process was repeated. The results showed that after being reused for 8 batches, the catalytic activity of the immobilized enzyme remained above 80% of the initial activity, demonstrating good operational stability and reusability.

[0066] The above embodiments demonstrate that the immobilized nucleoside enzyme mutant and its application method provided by the present invention can efficiently and greenly prepare guanine, and the catalyst is easy to recover and reuse, showing good prospects for industrial application.

Claims

1. An immobilized nucleoside enzyme mutant, characterized in that: It is prepared by covalently immobilizing the nucleoside enzyme mutant G178D / A202K, whose amino acid sequence is shown in SEQ ID NO:2, onto a magnetic Fe3O4 nanoparticle carrier that has been surface-aminated.

2. The immobilized nucleoside enzyme mutant according to claim 1, characterized in that: The magnetic Fe3O4 nanoparticle carrier is Fe3O4 nanoparticles modified with aminosilane reagent and rich in amino functional groups on the surface.

3. The immobilized nucleoside enzyme mutant according to claim 2, characterized in that: The aminosilane reagent is 3-aminopropyltriethoxysilane.

4. A method for preparing an immobilized nucleoside enzyme mutant according to any one of claims 1-3, characterized in that: Includes the following steps: S1. Preparation of magnetic Fe3O4 nanoparticles; S2. The Fe3O4 nanoparticles obtained in step S1 were modified by surface amylation using an aminosilane reagent to obtain an NH2-Fe3O4 support. S3. Disperse the NH2-Fe3O4 support in a buffer solution, add the cross-linking agent glutaraldehyde for activation, and obtain an aldehyde-activated magnetic support; S4. Mix the enzyme solution containing the nuclease mutant G178D / A202K with the activated vector obtained in step S3, and incubate to covalently immobilize the enzyme on the vector. S5. After incubation, the immobilized nucleoside enzyme mutant is obtained by separation and washing using a magnetic field.

5. The preparation method according to claim 4, characterized in that: In step S4, the incubation conditions are as follows: shake incubation for 4-16 hours in a buffer solution at 4-25°C and pH 7.0-8.

5.

6. A thermostable nucleoside enzyme mutant G178D / A202K, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

7. The use of the immobilized nucleoside enzyme mutant according to any one of claims 1-3, and the thermostable nucleoside enzyme mutant G178D / A202K according to claim 6, in catalyzing the hydrolysis of guanine nucleotides to guanine.

8. A method for preparing guanine using the immobilized nucleoside enzyme mutant according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Using water or buffer solution as the reaction medium, add the substrate guanine nucleotide and the immobilized nuclease mutant; (2) The reaction system of step (1) is subjected to hydrolysis at 50-70℃ with stirring until the substrate is completely converted; (3) After the reaction is completed, the immobilized nuclease mutant is separated from the reaction system and recovered using an external magnetic field; (4) The reaction liquid after separating the catalyst in step (3) is subjected to solid-liquid separation, the solid part is collected, washed and dried to obtain guanine product.

9. The method for preparing guanine according to claim 8, characterized in that: In step (1), the concentration of the substrate guanine nucleotide is 100-250 g / L, and the amount of the immobilized nuclease mutant added is 10-25 g / L based on the dry weight of the vector.

10. The method for preparing guanine according to claim 8, characterized in that: In step (2), the temperature of the catalytic reaction is 60-70℃ and the reaction time is 2-5 hours.

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