Process for preparing 5 '-guanylic acid by enzyme method

Abstract

The invention belongs to the technical field of biological medicines, and discloses a process for preparing 5 '-guanylic acid by an enzyme method. According to the process, 5 '-nucleotide phosphodiesterase modified by molecular imprinting is adopted as a catalyst, and RNA hydrolysis is catalyzed under the condition of controlling pH, temperature and substrate concentration; the 5 '-nucleotide phosphodiesterase modified by molecular imprinting is a composite catalytic unit, and the composite catalytic unit comprises a first layer which is a basic enzyme protein skeleton and contains a catalytic triad composed of histidine, aspartic acid and serine residues; the second layer is a substrate guide channel; and the third layer is a molecular imprinting template covalently anchored on lysine or cysteine residues at the inlet of the substrate guiding channel. According to the method, generation of 2 '-guanylic acid and 3'-guanylic acid byproducts is inhibited from the source, a high-purity product can be obtained only through single-step ion exchange, and solvent consumption and energy consumption are reduced; the overall yield is improved, and the waste of raw materials is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an enzymatic process for preparing 5'-guanosine monophosphate. Background Technology

[0002] 5′-Guanine acid, as an important flavor nucleotide, is widely used as a umami enhancer in the food industry and also holds key intermediate value in the pharmaceutical and biotechnology fields. Its preparation process has long relied on enzymatic hydrolysis of substrates derived from natural RNA. Among these, the catalytic system centered on 5′-nucleotide phosphodiesterase has become the mainstream industrial approach due to its advantages such as mild reaction conditions, environmental friendliness, and wide availability of substrates. This enzyme can specifically recognize and hydrolyze the 3′,5′-phosphodiester bonds between adjacent nucleotides in the RNA chain, theoretically generating 5′-monophosphate nucleosides, including the target product 5′-guanine acid. In the early stages of industrial application, this method was significantly superior to chemical synthesis routes, effectively avoiding side reactions and environmental pollution problems under strong acid and alkaline conditions.

[0003] However, although 5′-nucleotide phosphodiesterases are classified as “5′-specific” phosphodiesterases, their actual action on RNA substrates is not absolutely site-specific. This is because the enzyme's active site has limited spatial recognition ability for the 2′- and 3′-hydroxyl groups of the ribose ring, making it difficult to completely distinguish between isomers where the phosphodiester bond is attached to the 2′ or 3′ position of the ribose during catalysis. Consequently, when hydrolyzing guanine-rich RNA fragments, in addition to generating the target product 5′-guanosine nucleotide, positional isomers such as 2′-guanosine nucleotide and 3′-guanosine nucleotide are inevitably produced simultaneously as positional byproducts.

[0004] These byproducts are highly similar to the target product in terms of molecular weight, charge properties, and polarity, making subsequent separation and purification processes extremely difficult. Conventional ion exchange chromatography or crystallization methods often require multiple steps, consume large amounts of solvent, and result in product loss, which not only significantly increases production costs but also makes it difficult to meet the quality standards for high-purity products. Summary of the Invention

[0005] This invention provides an enzymatic process for preparing 5′-guanosine monophosphate, aiming to solve the technical problems in the prior art where the lack of site specificity of 5′-nucleotide phosphodiesterase for the hydrolysis of RNA substrates leads to the generation of 2′-guanosine monophosphate and 3′-guanosine monophosphate byproducts, resulting in difficult product separation, low yield, and high cost.

[0006] To achieve the above-mentioned objectives, this invention constructs a novel catalytic system based on an enzyme-substrate spatially guided matching mechanism. By introducing a molecularly imprinted template structure around the active site of 5′-nucleotide phosphodiesterase, the binding conformation of guanine nucleotide units is precisely defined. This forces the phosphodiester bond to enter the catalytic site only with a 5′ orientation during the substrate recognition stage, fundamentally blocking the non-target hydrolysis pathways of 2′- and 3′-phosphate bonds.

[0007] The core of the process described in this invention lies in the targeted functionalization modification of 5′-nucleotide phosphodiesterases from natural or recombinant sources to form a composite catalytic unit with spatially selective recognition capabilities. This composite catalytic unit consists of the following three structural levels: The first level is the basic enzyme protein backbone, whose amino acid sequence contains a complete catalytic triplet (usually a conserved sequence composed of histidine, aspartic and serine residues) to maintain basic hydrolytic activity against phosphodiester bonds. The second level is the substrate-guided channel, which consists of rigid aromatic amino acid side chains (such as tryptophan, tyrosine, or phenylalanine) introduced at specific sites on the enzyme surface, forming a narrow, elongated hydrophobic microcavity with π-π stacking ability. The third layer is the molecularly imprinted template layer. This template layer is not physically embedded outside the enzyme, but is fixed to the lysine or cysteine ​​residues at the entrance of the guide channel through covalent coupling. Its chemical structure precisely mimics the spatial arrangement of the guanine base and ribose ring of 5′-guanosine monophosphate, and steric hindrance groups are placed at the 2′- and 3′-hydroxyl positions of the template molecule, so that any substrate fragment that attempts to bind towards the catalytic center with the 2′- or 3′-phosphate bond cannot complete the binding effectively due to steric conflict.

[0008] The molecularly imprinted template is prepared and integrated into the enzyme molecule by the following steps: First, a bifunctional crosslinking agent is synthesized, with one end being an N-hydroxysuccinimide ester, which is used to undergo an amidation reaction with the ε-amino group of lysine on the enzyme surface; the other end is a guanine derivative containing a protecting group, the ribose portion of which is fully acetylated, and tert-butyldimethylsilyl groups are introduced at the 2′- and 3′- positions as temporary protecting groups, while a photolyzable o-nitrobenzyl phosphate group is attached at the 5′- position.

[0009] Subsequently, the cross-linking agent was incubated with purified 5′-nucleotide phosphodiesterase at pH 7.4 and 4°C for 12 hours to covalently link the N-hydroxysuccinimide ester end to specific lysine residues on the enzyme surface.

[0010] Next, under an inert atmosphere, tetrabutylammonium fluoride was used to remove the tert-butyldimethylsilyl protecting group, exposing the 2′- and 3′-hydroxyl groups. Immediately afterward, excess 2,2,6,6-tetramethylpiperidine-1-oxy radicals and sodium hypochlorite were added to a 1:1 volume ratio of dimethyl sulfoxide and water mixture to oxidize the 2′- and 3′-hydroxyl groups to carboxyl groups at 0°C, forming two negatively charged steric hindrance sites.

[0011] Finally, irradiation with 365nm ultraviolet light for 30 minutes cleaved the ortho-nitrobenzyl linkage, releasing the 5′-phosphate group, but retaining the entire guanine-ribose backbone as a permanent imprint template anchored to the enzyme surface.

[0012] After this treatment, the enzyme molecule forms a spatial screening window at the entrance of the guide channel that only allows the substrate to pass through with the 5′-phosphate bond facing inward, the guanine base embedded in the π-packing cavity, and no substituent interference at the 2′ / 3′ position.

[0013] In the actual catalytic reaction, the process described in this invention uses the modified 5′-nucleotide phosphodiesterase as a catalyst, yeast RNA or E. coli RNA hydrolysate as a substrate, and carries out the hydrolysis reaction under conditions of pH 6.8 to 7.2, temperature 30°C to 37°C, and substrate concentration of 50 g / L to 150 g / L. The reaction system also contains 10 mM to 50 mM magnesium ions as a cofactor, and 0.1% to 0.5% (mass fraction) of polyethylene glycol 6000 to stabilize the enzyme conformation.

[0014] During the reaction, when the guanylic acid residues in the RNA chain diffuse to the enzyme surface, they first undergo base complementarity recognition and hydrogen bond pairing with the molecularly imprinted template. If the phosphodiester bond is attached at the 5′ position, the entire nucleotide unit can smoothly slide into the guide channel and reach the catalytic center. If it is attached at the 2′ or 3′ position, the binding energy is significantly increased due to the electrostatic repulsion between the carboxyl group at the 2′ / 3′ position and the corresponding negative charge on the template, coupled with the steric hindrance effect. This prevents the formation of a stable enzyme-substrate complex, thus effectively excluding it from the catalytic cycle.

[0015] In another preferred embodiment of the present invention, the guanine base portion of the molecularly imprinted template is replaced with 8-azaguanine to enhance the hydrogen bond network strength with the substrate guanine. Simultaneously, a methoxy substituent is introduced at the 4′-position of the ribosome to further restrict the conformational inversion freedom of the substrate ribosome. After this variant template is integrated into the enzyme molecule via the same covalent coupling strategy, it maintains over 95.2% 5′-guanylic acid selectivity in the hydrolysis of low-G-content RNA (G content ≤20%) under the same reaction conditions, indicating that the spatial steering mechanism described in this invention has good adaptability to different substrate compositions.

[0016] The process described in this invention also includes an integrated reaction-separation coupling system to further improve product purity and yield. This system consists of a continuous stirred tank reactor, an online pH control module, a membrane separation unit, and a gradient elution ion exchange column connected in series. After hydrolysis in the continuous stirred tank reactor, the reaction solution is immediately filtered through a 0.22 μm microporous membrane to remove enzyme proteins. The filtrate then enters a nanofiltration membrane module with a molecular weight cutoff of 300 Da, retaining unreacted large RNA fragments and returning them to the reactor. The permeate is then introduced into a strongly basic anion exchange resin column (the matrix is ​​cross-linked polystyrene-divinylbenzene, and the functional groups are quaternary ammonium groups). Due to the spatial configuration difference between 2′-guanosine and 3′-guanosine, their binding constants with the resin are slightly lower than those of 5′-guanosine. When eluting with a sodium chloride gradient (0.05 M to 0.3 M), the byproducts are eluted before the main product, thus achieving one-step separation.

[0017] The technical parameters of the process described in this invention also include: the density of the molecularly imprinted template is controlled at 1.8 to 2.2 template units per enzyme molecule. Too high a density will lead to increased substrate diffusion resistance, while too low a density will prevent the formation of an effective spatial sieving effect; the reaction time is controlled at 4 to 8 hours to avoid enzyme autohydrolysis or product degradation caused by prolonged reaction; and 0.05 mM to 0.2 mM of dithiothreitol is added to the reaction system to maintain the reduced state of cysteine ​​residues at the enzyme active site and prevent oxidative inactivation.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. It inhibits the formation of 2′-guanosine monophosphate and 3′-guanosine monophosphate byproducts at the source, simplifies the subsequent purification process, and only requires a single ion exchange to obtain high-purity products, reducing solvent consumption and energy consumption; it also improves the overall yield and reduces raw material waste. 2. This space-oriented design concept is universal and can be extended to the modification of other nucleotide phosphodiesterases, providing a new technical paradigm for highly selective biocatalysis.

[0019] 3. The molecularly imprinted template described in this invention is not a traditional polymer imprinted material, but rather a recognition site with atomic-level precision constructed in situ on the surface of the enzyme molecule through precise organic synthesis and protein chemical modification. This template is covalently linked to the enzyme protein, exhibiting high stability and showing no significant detachment or inactivation. Attached Figure Description

[0020] Figure 1 This is a bar chart comparing the test data of 5′-guanosine monophosphate selectivity, product purity, and total yield of the examples and comparative examples; Figure 2 This is a line graph comparing the test data of the total content of by-products and the reduction rate of solvent consumption in the examples and comparative examples. Detailed Implementation

[0021] This invention provides an enzymatic process for the preparation of 5′-guanosine monophosphate (GMP), aiming to solve the technical problems in existing technologies where the lack of site specificity of 5′-nucleotide phosphodiesterase for RNA substrate hydrolysis leads to the generation of 2′-guanosine monophosphate and 3′-guanosine monophosphate byproducts, resulting in difficult product separation, low yield, and high cost. To achieve the above objective, this invention constructs a novel catalytic system based on an enzyme-substrate spatially guided matching mechanism. By introducing a molecularly imprinted template structure around the active site of 5′-nucleotide phosphodiesterase, the binding conformation of guanine nucleotide units is precisely defined. This forces the phosphodiester bond to enter the catalytic site only with a 5′ orientation during the substrate recognition stage, fundamentally blocking the non-target hydrolysis pathways of 2′- and 3′-phosphate bonds.

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0023] Example 1: Enzyme source: Bacillus subtilis.

[0024] Molecular imprinted template: Guanine-ribose backbone with unreplaced bases and ribose ring substituents.

[0025] Template density: 2.0 template units anchored per enzyme molecule.

[0026] Substrate: Yeast RNA, G content 38%.

[0027] Reaction conditions: pH 7.0, temperature 35℃, substrate concentration 100g / L, magnesium ions 30mM, polyethylene glycol 6000 (mass fraction) 0.3%, dithiothreitol 0.1mM, reaction time 6 hours.

[0028] Separation system: Continuous stirred tank reactor, online pH control module, membrane separation unit, gradient elution, ion exchange column in series Example 2: Enzyme source: Same as Example 1.

[0029] Molecular imprinting template: Guanine base replacement with 8-azaguanine ribose ring, and introduction of a methoxy substituent at the 4′ position. Template density: 2.1 template units anchored per enzyme molecule.

[0030] Substrate: Escherichia coli RNA, G content 19%.

[0031] Reaction conditions: pH 7.1, temperature 36℃, substrate concentration 80g / L, magnesium ions 25mM, polyethylene glycol 6000 (mass fraction) 0.2%, dithiothreitol 0.08mM, reaction time 7 hours.

[0032] Separation system: Same as in Example 1.

[0033] Example 3: Enzyme source: Same as Example 1.

[0034] Molecular imprint template: Same as in Example 1.

[0035] Template density: 2.0 template units anchored per enzyme molecule.

[0036] Substrate: Same as in Example 1.

[0037] Reaction conditions: pH 6.8, temperature 35℃, substrate concentration 100g / L, magnesium ions 30mM, polyethylene glycol 6000 (mass fraction) 0.3%, dithiothreitol 0.1mM, reaction time 6.5 hours.

[0038] Separation system: Same as in Example 1.

[0039] Example 4: Enzyme source: Same as Example 1.

[0040] Molecular imprint template: Same as in Example 1.

[0041] Template density: 2.0 template units anchored per enzyme molecule.

[0042] Substrate: Same as in Example 1.

[0043] Reaction conditions: pH 7.0, temperature 30℃, substrate concentration 100g / L, magnesium ions 30mM, polyethylene glycol 6000 (mass fraction) 0.3%, dithiothreitol 0.1mM, reaction time 7.5 hours.

[0044] Separation system: Same as in Example 1.

[0045] Example 5: Enzyme source: Same as Example 1.

[0046] Molecular imprint template: Same as in Example 1.

[0047] Template density: 2.2 template units anchored per enzyme molecule.

[0048] Substrate: Same as in Example 1.

[0049] Reaction conditions: pH 7.0, temperature 35℃, substrate concentration 150g / L, magnesium ions 40mM, polyethylene glycol 6000 (mass fraction) 0.5%, dithiothreitol 0.15mM, reaction time 8 hours.

[0050] Separation system: Same as in Example 1.

[0051] Example 6: Enzyme source: Same as Example 1.

[0052] Molecular imprint template: Same as in Example 1.

[0053] Template density: 2.0 template units anchored per enzyme molecule.

[0054] Substrate: Same as in Example 1.

[0055] Reaction conditions: pH 7.0, temperature 35℃, substrate concentration 100g / L, magnesium ions 50mM, polyethylene glycol 6000 (mass fraction) 0.3%, dithiothreitol 0.1mM, reaction time 5.5 hours.

[0056] Separation system: Same as in Example 1.

[0057] Example 7: Enzyme source: Same as Example 1.

[0058] Molecular imprint template: Same as in Example 1.

[0059] Template density: 1.8 template units are anchored per enzyme molecule.

[0060] Substrate: Same as in Example 1.

[0061] Reaction conditions: pH 7.0, temperature 35℃, substrate concentration 100g / L, magnesium ions 30mM, polyethylene glycol 6000 (mass fraction) 0.3%, dithiothreitol 0.1mM, reaction time 6 hours.

[0062] Separation system: Same as in Example 1.

[0063] Comparative Example 1: Enzyme source: Bacillus subtilis, without molecular imprinting modification.

[0064] Substrate: Same as in Example 1.

[0065] Reaction conditions: Same as in Example 1.

[0066] Separation system: Same as in Example 1.

[0067] Comparative Example 2: Process route: Ribose and guanine are condensed by strong acid catalysis and then separated and purified by multiple phosphorylation steps.

[0068] Substrate: Chemically pure ribose and chemically pure guanine.

[0069] Reaction conditions: concentrated hydrochloric acid catalysis, temperature 80℃, reaction time 12 hours, multi-step recrystallization separation.

[0070] Test method: 5′-Guanine acid selectivity: The selectivity was calculated by detecting the peak area ratio of 5′-guanine acid to total nucleotide products in the reaction solution using high performance liquid chromatography.

[0071] Total content of by-products: The sum of the peak areas of 2′-guanosine monophosphate and 3′-guanosine monophosphate were calculated by high performance liquid chromatography to determine their proportion in the total nucleotide products.

[0072] Product purity: After purification by ion exchange chromatography, the purity is verified by high performance liquid chromatography and expressed as the ratio of the 5′-guanylic acid peak area to the total peak area.

[0073] Overall yield: The ratio of the actual yield of 5′-guanosine monophosphate product to the theoretical yield of guanine nucleotides in the substrate RNA.

[0074] Solvent consumption reduction percentage: The reduction percentage is calculated by comparing the total solvent consumption of each scheme with that of the traditional multi-step purification process.

[0075] Test data comparison table:

[0076] Molecular imprinting modification is the core of improving selectivity. Examples 1-7 all use molecular imprinting modification. The 5′-guanosine monophosphate selectivity of the enzymes is over 95%, and the total content of by-products is less than 5%. In contrast, the unmodified comparative example 1 has a selectivity of only 76.4% and a by-product content as high as 23.6%. This shows that the molecular imprinted template blocks the hydrolysis of non-target sites from the source through spatial guidance and repulsion.

[0077] Example 2: Template variant adapted to substrates with low G content. The template with 8-azaguanine and 4′-methoxy substitution still maintained 95.2% selectivity for E. coli RNA with a G content of 19%, indicating that the template variant enhanced the interaction with the substrate and broadened the substrate applicability.

[0078] The reaction conditions affect the process effect. Adjustments to parameters such as pH, temperature, and substrate concentration cause slight fluctuations in the selective yields of Examples 3-7, but all remain at a high level. Among them, pH 6.8-7.2 and temperature 30-37℃ are the optimal ranges. Higher substrate concentrations require higher concentrations of magnesium ions and polyethylene glycol 6000 to ensure enzyme activity and stability.

[0079] The process advantages of this invention are significant. Compared with the traditional chemical synthesis comparative example 2, the total yield of Examples 1-7 is 26.8-39 percentage points higher, solvent consumption is reduced by 57%-60%, and the product purity is higher. Compared with the unmodified enzyme process, the yield is increased by 19.8-22 percentage points, solvent consumption is significantly reduced, and it meets pharmaceutical grade standards.

[0080] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for the enzymatic preparation of 5′-guanylic acid, characterized in that, A molecularly imprinted 5′-nucleotide phosphodiesterase was used as a catalyst to catalyze RNA hydrolysis under controlled pH, temperature, and substrate concentration conditions. The molecularly imprinted 5′-nucleotide phosphodiesterase is a composite catalytic unit, which comprises: The first level is the basic enzyme protein backbone, which contains a catalytic triplet composed of histidine, aspartic, and serine residues. The second level is the substrate-guided channel, which consists of rigid aromatic amino acid side chains introduced at specific sites on the enzyme surface, forming a hydrophobic microcavity with π-π stacking ability. The third level is a molecularly imprinted template covalently anchored to a lysine or cysteine ​​residue at the entrance of the substrate guiding channel. The molecularly imprinted template simulates the spatial arrangement of the guanine base and ribose ring of 5′-guanosine monophosphate and has negatively charged steric repulsion groups at its 2′- and 3′- positions to force the substrate to bind only with the 5′-phosphate bond toward the catalytic center.

2. The process for preparing 5′-guanosine monophosphate by enzymatic method according to claim 1, characterized in that, The basic enzyme protein backbone is derived from Bacillus subtilis.

3. The process for preparing 5′-guanosine monophosphate by enzymatic method according to claim 2, characterized in that, The basic enzyme protein backbone was subjected to site-directed mutagenesis, replacing the glutamic acid at position 187 with lysine to introduce an additional coupling site at the substrate-guided channel entrance.

4. The process for preparing 5′-guanosine monophosphate by enzymatic method according to claim 1, characterized in that, The rigid aromatic amino acid side chain includes at least one of tryptophan, tyrosine, or phenylalanine.

5. The process for preparing 5′-guanosine monophosphate by enzymatic method according to claim 1, characterized in that, The molecularly imprinted template is covalently linked to the enzyme surface via a bifunctional cross-linking agent. One end of the bifunctional cross-linking agent is an N-hydroxysuccinimide ester, and the other end is a guanine ribose derivative that has been fully acetylated and has 2′- and 3′-tert-butyldimethylsilyl protecting groups and a 5′-o-nitrobenzyl phosphate group.

6. The process for the enzymatic preparation of 5′-guanosine monophosphate according to claim 5, characterized in that, The preparation of the molecularly imprinted template includes the following steps: The bifunctional cross-linking agent and enzyme were reacted at pH 7.4 and 4°C for 12 hours to complete the coupling. Subsequently, the protecting groups at the 2′ and 3′ positions were removed using tetrabutylammonium fluoride; The exposed 2′- and 3′-hydroxyl groups are then oxidized to carboxyl groups using a 2,2,6,6-tetramethylpiperidine-1-oxygen radical / sodium hypochlorite system. Finally, the o-nitrobenzyl linkage was broken by 365nm ultraviolet light irradiation, releasing the 5′-phosphate group and retaining the guanine-ribose backbone as a permanent template.

7. The process for the enzymatic preparation of 5′-guanosine monophosphate according to claim 1, characterized in that, Each molecule of the composite catalytic unit is anchored with 1.8 to 2.2 molecularly imprinted templates.

8. The process for the enzymatic preparation of 5′-guanosine monophosphate according to claim 1, characterized in that, The catalytic reaction system also contains 10 mM to 50 mM magnesium ions, 0.1% to 0.5% by mass of polyethylene glycol 6000, and 0.05 mM to 0.2 mM dithiothreitol.

9. The process for the enzymatic preparation of 5′-guanosine monophosphate according to claim 1, characterized in that, The RNA substrate is yeast RNA or E. coli RNA hydrolysate.

10. The process for the enzymatic preparation of 5′-guanosine monophosphate according to claim 1, characterized in that, The guanine base in the molecularly imprinted template is replaced with 8-azaguanine, and a methoxy substituent is present at the 4′-position of the ribosome.

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