A cytidine deaminase continuous flow catalytic system based on t4 bacteriophage and agarose microsphere composite material and a construction method thereof

Abstract

The application provides a cytidine deaminase continuous flow catalytic system based on a T4 bacteriophage and an agarose microsphere composite material and a construction method thereof, so as to solve the technical problems of the existing cytidine deaminase enzyme catalytic system, such as large enzyme activity loss, uncontrollable enzyme molecular immobilization orientation, insufficient continuous flow fixed bed adaptability, and incomplete performance evaluation dimension, and the like.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and immobilized enzyme continuous flow reaction technology, specifically relating to a cytidine deaminase continuous flow catalytic system based on T4 phage and agarose microsphere composite material and its construction method. Background Technology

[0002] Cytidine deaminases catalyze the deamination of cytidine and related nucleoside substrates, holding significant value in the green biosynthesis of nucleoside compounds, pyrimidine intermediates, and related pharmaceuticals and fine chemicals. Compared to traditional chemical synthesis methods, enzyme-catalyzed processes typically offer advantages such as milder reaction conditions, higher substrate selectivity, fewer side reactions, and lower environmental impact, thus attracting widespread attention in green manufacturing and biocatalytic synthesis. Particularly in the preparation of nucleoside compounds, enzymatic routes can reduce the use of high temperatures, high pressures, and toxic reagents, demonstrating promising industrial application prospects.

[0003] However, in practical applications, free enzyme systems still face several limitations. First, free enzymes are susceptible to changes in temperature, pH, shear force, and the substrate or product environment within the reaction system, leading to decreased or even inactivated enzyme activity. Their stability often fails to meet the requirements of continuous production and process scale-up. Second, free enzymes dispersed in the reaction solution are difficult to recover and reuse efficiently after the reaction, often increasing catalyst costs and hindering process economics. Third, free enzyme systems are prone to loss with the fluid during continuous operation, making it difficult to directly construct a stable fixed-bed catalytic phase, thus resulting in poor compatibility with continuous flow fixed-bed reactors. These issues, to some extent, restrict the further application of cytidine deaminase-based enzyme catalytic systems in industrial green synthesis.

[0004] To improve enzyme stability, recyclability, and process controllability, enzyme immobilization technology has become an important technical route in the field of biocatalysis. By immobilizing enzymes on or inside an insoluble support, enzyme tolerance and reusability can be improved to a certain extent, and it is beneficial for reactor loading and continuous operation. Existing immobilization methods mainly include physical adsorption, embedding, cross-linking, and chemical covalent immobilization. Among them, chemical immobilization methods are widely used in the construction of continuous flow reaction systems due to their strong binding and minimal enzyme loss.

[0005] However, traditional direct chemical immobilization methods typically involve directly covalently linking enzyme molecules to the support surface. While this approach can improve enzyme immobilization strength, it also easily introduces new problems. Because the distribution of reactive groups on the enzyme molecule surface is random, direct chemical coupling often leads to disordered orientation of the enzyme on the support surface. This results in unfavorable orientation of the active site, hindered substrate access, or local conformational damage, ultimately causing a significant decrease in catalytic activity after immobilization. Especially under high-density immobilization conditions, spatial crowding and localized stacking may occur between enzyme molecules, further weakening mass transfer efficiency and activity retention. Therefore, how to ensure immobilization strength and continuous flow applicability while simultaneously optimizing the ordered arrangement of enzyme molecules and the active microenvironment is a key issue in the design of immobilized enzyme materials.

[0006] On the other hand, continuous flow fixed-bed reactors have become an important direction for enhancing biocatalytic processes due to their advantages such as continuous feed, short downtime, high process stability, ease of scale-up, and ability to improve space-time yield. Compared to traditional batch reactions, continuous flow reactors can achieve more efficient and controllable catalytic processes by stably controlling parameters such as reaction time, flow rate, substrate concentration, and temperature. However, to achieve efficient continuous flow operation, fixed-bed catalytic materials not only need to have high enzyme loading and catalytic activity, but also low bed flow resistance, good mechanical stability, small pressure drop changes, and high long-term operational stability. Although some existing immobilized materials can achieve enzyme loading, they still suffer from problems such as high flow resistance, limited mass transfer, and rapid activity decay during operation under continuous flow conditions, making it difficult to simultaneously meet the dual requirements of fixed-bed engineering applications and efficient biocatalysis.

[0007] The T4 phage capsid surface exhibits unique advantages in nanobiomaterials and enzyme assembly due to its regular arrangement, well-defined sites, programmable modification capabilities, and ability to perform specific recognition and assembly. In particular, the Soc-related binding sites on the T4 phage surface can specifically recognize fusion proteins carrying Soc tags, providing a novel strategy for the targeted loading of enzyme molecules, distinct from random chemical fixation. If T4 phage is introduced as a nano-scaffold into a microsphere immobilization system, it is possible to construct a hierarchical enzyme immobilization interface on the surface of the macroscopic microsphere carrier. This involves first stably coupling the T4 phage to the microsphere surface, and then using the Soc tag to achieve the targeted self-assembly of functional enzymes on the phage surface. This strategy is expected to improve the orientation distribution and local microenvironment of enzyme molecules while maintaining the performance of fixed-bed loading, reducing the adverse effects of direct chemical fixation on the enzyme active site and conformation, thus achieving a balance between high activity retention, high loading capacity, and continuous flow stability.

[0008] Currently, research on constructing a multi-level composite immobilization system of cytidine deaminase CD1.3 (enzyme-T4 phage-microsphere) and further applying it to continuous flow fixed-bed catalysis and system performance characterization is still relatively limited. In particular, in systems using carboxylated agarose microspheres as the fixed-bed substrate, T4 phage as the nano-intermediate scaffold, and CD1.3-Soc fusion enzyme as the functional catalytic unit, how to effectively construct composite catalytic materials and systematically evaluate their continuous flow catalytic performance in terms of flow resistance pressure, catalytic activity, kinetic behavior, flow rate response, space-time yield, recycling stability, and storage stability still lacks comprehensive technical solutions and application data. Summary of the Invention

[0009] To address the problems of poor stability of free enzymes, difficulty in recycling, insufficient adaptability to continuous production, and significant enzyme activity loss and limited bed mass transfer performance caused by traditional direct immobilization methods in the background technology, this invention proposes a continuous flow catalytic system for cytidine deaminase based on T4 phage and agarose microsphere composite materials and its construction method. This aims to solve the technical problems of significant enzyme activity loss, uncontrollable enzyme molecule immobilization orientation, insufficient adaptability to continuous flow immobilized beds, and incomplete performance evaluation dimensions in existing cytidine deaminase catalytic systems using traditional direct immobilization methods.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The first objective of this invention is to propose a continuous flow catalytic system for cytidine deaminase based on a composite material of T4 phage and agarose microspheres. T4 phage nanoscaffolds are introduced onto the surface of carboxylated agarose microspheres, and the specific recognition between the Soc tag and the T4 phage capsid surface sites is utilized to achieve the directional self-assembly of CD1.3-Soc enzyme on a fixed-bed surface, constructing a continuous flow reactor catalytic platform that combines fixed-bed engineering applicability with high catalytic activity retention. Simultaneously, the enzyme loading process, continuous flow catalytic activity, flow resistance pressure, kinetic behavior, flow rate response, space-time yield, cycling stability, and storage stability of this platform are systematically characterized, providing comprehensive technical support for its application in continuous flow biocatalysis.

[0012] The second objective of this invention is to propose a method for constructing a continuous flow catalytic system for cytidine deaminase based on a composite material of T4 bacteriophage and agarose microspheres. This invention first uses carboxylated agarose microspheres as the fixed-bed substrate material and constructs the main body of a continuous flow fixed-bed reactor by packing a micro-medium-pressure chromatography column. The carboxylated agarose microspheres serve both as the macroscopic carrier of the immobilized enzyme and as the bed packing material of the continuous flow reactor, providing a stable flow path structure and good engineering packing performance.

[0013] After the fixed bed was constructed, the carboxyl groups on the surface of the carboxylated agarose microspheres were activated using EDC·HCl. T4 phage was then introduced into the fixed bed system, covalently immobilizing the T4 phage onto the microsphere surface to form a phage-microsphere composite material. In this invention, the T4 phage serves as a nano-scaffold, providing regular and identifiable surface sites for subsequent enzyme loading.

[0014] After phage coupling, the resulting phage-microsphere composite material was blocked to reduce non-specific adsorption. Subsequently, CD1.3-Soc enzyme solution was introduced into the system, allowing the CD1.3-Soc enzyme to complete directional self-assembly through the specific recognition between the Soc tag and the T4 phage capsid surface site, thereby obtaining the enzyme-T4 phage-microsphere composite catalytic material.

[0015] The enzyme-T4 phage-microsphere composite catalytic material was loaded into a continuous flow fixed-bed reactor, and a cytidine substrate solution was continuously introduced under the drive of a constant flow pump. A steady-state continuous flow catalytic reaction was carried out under set temperature, pH and flow rate conditions, thereby constructing a CD1.3 continuous flow reactor catalytic platform based on the enzyme-T4 phage-microsphere composite material.

[0016] The third objective of this invention is to propose a performance characterization method for a continuous flow catalytic system of cytidine deaminase based on a composite material of T4 bacteriophage and agarose microspheres, the method being as follows:

[0017] After completing the construction of the continuous flow reactor catalytic platform, the construction of the composite catalytic material and the enzyme loading process were first analyzed. Specifically, SDS-PAGE was used to detect the enzyme loading process.

[0018] After confirming enzyme loading, the effluent from the continuous flow catalytic reaction was analyzed. The catalytic function of the continuous flow reactor catalytic platform was characterized by analyzing the chromatographic peak changes of the cytidine substrate and N-hydroxycytidine product using HPLC.

[0019] Based on the characterization of catalytic function, the enzyme-T4 phage-microsphere composite catalytic system constructed in this invention was further compared with the free CD1.3-Soc enzyme system and the microsphere directly chemically immobilized enzyme system. The improvement effect of the composite immobilization strategy of this invention on the maintenance of catalytic activity was evaluated by conversion rate analysis.

[0020] Subsequently, pre-column pressure tests were conducted on the original carboxy-agarose microsphere immobilized bed, the phage-microsphere composite immobilized bed, and the enzyme-T4 phage-microsphere composite immobilized bed. By comparing the changes in flow resistance under different immobilized bed conditions, the fluid permeability and bed stability of the platform were characterized.

[0021] By setting different substrate concentrations and flow rates, the kinetics of the continuous flow catalytic platform were fitted and the flow rate response was analyzed. Specifically, the Lilly-Hornby model was used to fit the apparent kinetic parameters of the system, and the effect of flow rate changes on the catalytic performance of the system was evaluated by comparing the substrate conversion and catalytic efficiency under different flow rate conditions.

[0022] Based on this, the space-time yield of the continuous flow catalytic platform of the present invention is characterized by calculating the product generation per unit time and per unit reactor volume, and compared with the batch catalytic system of free enzymes to evaluate the process enhancement effect of the platform.

[0023] Finally, the activity retention capacity of the continuous flow catalytic platform under low-temperature storage conditions and during multiple rounds of repeated use were characterized by storage stability test and recycling stability test, respectively, in order to evaluate its long-term operational suitability.

[0024] Compared with existing technologies, the above technical solution can achieve the following beneficial effects:

[0025] This invention introduces T4 phage as a nano-scaffold on the surface of carboxyagarose microspheres and utilizes Soc tags to achieve the directed self-assembly of CD1.3-Soc enzymes. This effectively improves the activity loss problems caused by random orientation of enzyme molecules and masking of active sites in traditional direct chemical immobilization methods. Experimental results show that the relative catalytic activity of the enzyme-T4 phage-microsphere composite catalytic material constructed in this invention reaches 39%, significantly higher than the 17% of enzyme materials directly immobilized by microspheres, and close to the 41% of the free CD1.3-Soc enzyme system. This indicates that this invention can maintain the catalytic function of enzyme molecules while maintaining the immobilization strength.

[0026] The enzyme-T4 phage-microsphere composite fixed bed constructed in this invention combines the macroscopic packing characteristics of microsphere fixed beds with the surface assembly advantages of phage nanoscaffolds. It maintains a low in-column pressure in a continuous flow fixed bed reactor, indicating good fluid permeability and bed stability. These results demonstrate that after introducing the T4 phage nanoscaffold and completing the self-assembly of the CD1.3-Soc enzyme, the fixed bed system can still meet the requirements of low flow resistance and stable mass transfer for continuous flow operation, exhibiting good engineering adaptability.

[0027] This invention utilizes a constructed composite fixed bed for continuous flow catalysis of cytidine, significantly improving product output per unit time and unit reactor volume, demonstrating a clear process intensification effect. Experimental results show that the space-time yield of the continuous flow catalytic system of this invention reaches 2112.75 mM·L⁻¹·h⁻¹, significantly higher than the 310.48 mM·L⁻¹·h⁻¹ of the free enzyme batch catalytic system, indicating that the continuous flow reactor catalytic platform established in this invention has a significant advantage in production efficiency.

[0028] This invention establishes a systematic performance characterization scheme that includes enzyme loading analysis, continuous flow catalytic activity detection, flow resistance and pressure testing, kinetic fitting, flow rate response analysis, space-time yield evaluation, cycle life stability testing, and storage stability testing. Compared to existing technologies that only focus on immobilized enzyme activity or single catalytic results, this invention provides a systematic analysis of the continuous flow catalytic platform from multiple dimensions, including material construction, reactor operation, and long-term performance, making the performance evaluation of this system more complete, comprehensive, and reliable.

[0029] The continuous flow catalytic platform constructed in this invention exhibits both good reusability and low-temperature storage stability, indicating that the enzyme-T4 phage-microsphere composite catalytic material retains good activity even under multiple rounds of continuous flow operation and storage conditions. Therefore, this invention is not only applicable to continuous flow biocatalysis research of CD1.3 enzymes, but also provides a feasible technical route and application basis for the design of immobilized materials and the development of continuous flow processes in the green continuous synthesis of nucleoside compounds. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the construction method of the CD1.3 continuous flow catalytic platform based on enzyme-T4 phage-microsphere composite material.

[0031] In this diagram, A is a schematic diagram of the formation of a phage-microsphere composite material by coupling carboxylated agarose microspheres with T4 phage after EDC activation; B is a schematic diagram of the formation of an enzyme-T4 phage-microsphere composite catalytic material by CD1.3-Soc enzyme specifically recognizing the surface site of T4 phage through the Soc tag after the phage-microsphere composite material is blocked by BSA; C is a schematic diagram of the composite catalytic material being loaded into a continuous flow fixed bed reactor and used for continuous flow catalytic conversion of cytidine.

[0032] Figure 2 The figure shows the performance analysis of the CD1.3 continuous flow catalytic platform based on enzyme-T4 phage-microsphere composite material.

[0033] The following graphs are presented: SDS-PAGE protein electrophoresis (A) shows the analysis of the construction and enzyme loading process of the composite catalytic material; HPLC chromatogram (B) shows the detection results of cytidine, N(4)-hydroxycytidine, and continuous flow reaction samples; bar chart (C) shows the conversion rate comparison results of different catalytic systems; bar chart (D) shows the column inlet pressure comparison results of different fixed-bed systems; scatter plot and fitting curve (E) shows the Lilly-Hornby kinetic fitting results of the continuous flow fixed-bed reactor under different flow rate conditions; line graph (F) shows the changes in substrate conversion rate and catalytic efficiency under different flow rate conditions; bar chart (G) shows the space-time yield comparison results of the continuous flow catalytic system and the free enzyme batch catalytic system; bar chart (H) shows the relative activity results of the composite catalytic material under different storage times; and bar chart (I) shows the relative activity results of the composite catalytic material under different number of cycles. The error bar represents the standard error of three parallel replicate experiments. Detailed Implementation

[0034] The following is in conjunction with the appendix Figure 1 and 2 The present invention will be further illustrated by the following examples:

[0035] Figure 1 As shown, a continuous flow catalytic system for cytidine deaminase based on a composite material of T4 phage and agarose microspheres is presented. T4 phage nanoscaffolds are introduced onto the surface of carboxylated agarose microspheres, and the specific recognition between the Soc tag and the T4 phage capsid surface sites enables the directional self-assembly of CD1.3-Soc enzyme on a fixed-bed surface. This constructs a continuous flow reactor catalytic platform that combines fixed-bed engineering applicability with high catalytic activity retention. Furthermore, the enzyme loading process, continuous flow catalytic activity, flow resistance pressure, kinetic behavior, flow rate response, space-time yield, cycling stability, and storage stability of this platform are systematically characterized, providing comprehensive technical support for its application in continuous flow biocatalysis.

[0036] Its construction method is as follows:

[0037] This invention first uses carboxylated agarose microspheres as the fixed-bed substrate material and constructs the main body of a continuous flow fixed-bed reactor by packing micro-medium-pressure chromatography columns. The carboxylated agarose microspheres serve both as macroscopic carriers for immobilized enzymes and as bed packing material for the continuous flow reactor, providing a stable flow path structure and good engineering packing performance.

[0038] After the fixed bed was constructed, the carboxyl groups on the surface of the carboxylated agarose microspheres were activated using EDC·HCl. T4 phage was then introduced into the fixed bed system, covalently immobilizing the T4 phage onto the microsphere surface to form a phage-microsphere composite material. In this invention, the T4 phage serves as a nano-scaffold, providing regular and identifiable surface sites for subsequent enzyme loading.

[0039] After phage coupling, the resulting phage-microsphere composite material was blocked to reduce non-specific adsorption. Subsequently, CD1.3-Soc enzyme solution was introduced into the system, allowing the CD1.3-Soc enzyme to complete directional self-assembly through the specific recognition between the Soc tag and the T4 phage capsid surface site, thereby obtaining the enzyme-T4 phage-microsphere composite catalytic material.

[0040] The enzyme-T4 phage-microsphere composite catalytic material was loaded into a continuous flow fixed-bed reactor, and a cytidine substrate solution was continuously introduced under the drive of a constant flow pump. A steady-state continuous flow catalytic reaction was carried out under set temperature, pH and flow rate conditions, thereby constructing a CD1.3 continuous flow reactor catalytic platform based on the enzyme-T4 phage-microsphere composite material.

[0041] In a preferred embodiment of the present invention, the technical process includes the following steps:

[0042] (1) Fixed bed loading: Carboxy agarose microspheres that have been washed with ultrapure water and equilibrated with reaction buffer are loaded into a micro medium-pressure chromatography column by gravity sedimentation to form a 1 mL fixed bed, and the upper and lower sieve plates are installed and the inlet and outlet liquid flow paths are connected.

[0043] (2) Pretreatment of the flow path: The flow path is vented with ultrapure water and then continuously flushed with reaction buffer at a flow rate of 0.6 mL / min for about 10 column volumes to bring the fixed bed flow path to a stable equilibrium state.

[0044] (3) Phage coupling: 10 mg / mL EDC·HCl solution was introduced into the packed fixed bed and circulated for 45 min to activate the carboxyl groups on the surface of the carboxyl agarose microspheres; after activation, T4 phage solution was introduced for cyclic coupling, and the column bed was washed with reaction buffer after coupling to remove uncoupled free phages.

[0045] (4) Blocking treatment: The phage-microsphere composite fixed bed was introduced into the blocking solution containing 2 mg / mL BSA and circulated for 6 h to reduce non-specific adsorption during the subsequent enzyme loading process; after the blocking was completed, the fixed bed was rinsed with buffer.

[0046] (5) Enzyme self-assembly: The fixed bed was switched to PI-Mg buffer for equilibration, and then 2.0 mg of CD1.3-Soc enzyme solution was injected into the system and circulated for 1 h, so that it could complete directional self-assembly through the specific recognition between the Soc tag and the T4 phage capsid surface site, thus obtaining the enzyme-T4 phage-microsphere composite fixed bed reactor.

[0047] (6) Continuous flow catalysis: Cytidine is used as a substrate and is continuously introduced into a fixed bed reactor under set temperature, pH and flow rate conditions to carry out catalytic reaction; after the system reaches steady state, the effluent sample is collected.

[0048] (7) Sample processing and detection: The effluent from the continuous flow reactor was terminated with methanol, diluted with formic acid water and filtered through a filter membrane. The substrate and product were then detected and analyzed by HPLC.

[0049] (8) Platform performance characterization: The enzyme loading process was characterized by SDS-PAGE and BSA standard curves, respectively; the fixed bed flow resistance was characterized by pre-column pressure test; the kinetic fitting and flow rate response analysis were performed by substrate concentration gradient and flow rate gradient experiments combined with the Lilly-Hornby model; the space-time yield was calculated by product quantification results; and the cycling stability and storage stability were characterized by multiple rounds of repeated catalysis experiments and activity tests after low-temperature storage, respectively.

[0050] The constructed system achieved high enzyme activity retention and significantly better process enhancement than the free enzyme batch system while maintaining low flow resistance. Figure 2 As shown, the results indicate that the self-assembled composite material group achieved a cytidine conversion rate of 39% after 10 min of reaction at room temperature, significantly higher than the 17% of the direct chemical immobilization group and close to the 41% of the free enzyme group. The column inlet pressure of the fixed bed at a flow rate of 1 mL / min increased only from 0.21 Bar of the original microspheres to 0.28 Bar after the composite material was packed. The space-time yield of the continuous flow system was 2112.75 mM·L⁻¹·h⁻¹, which is 6.8 times that of the 310.48 mM·L⁻¹·h⁻¹ of the free enzyme batch system. The reactor maintained more than 80% of its relative activity after 10 reuses and still maintained more than 80% of its initial activity after storage at 4℃ for 14 days. This invention provides a reproducible technical solution for the construction and performance evaluation of immobilized continuous flow biocatalytic systems based on bacteriophage nanoscaffolds.

[0051] The construction and characterization methods of the system will be described one by one through examples below:

[0052] Specific Implementation Example 1: Construction of a Continuous Flow Reactor Catalytic Platform

[0053] 1.1 Preparation of the fixed bed

[0054] A micro-medium-pressure chromatography column was selected as the main body of the continuous flow fixed-bed reactor. The carboxyagarose microspheres were thoroughly washed with ultrapure water to remove the storage solution and any impurities that might be present, and then pre-equilibrated with reaction buffer to place the microspheres in a buffer environment suitable for the subsequent coupling reaction.

[0055] Pretreated carboxyagarose microspheres were slowly packed into a micro-medium-pressure chromatography column using gravity sedimentation, with a packing volume controlled at 1 mL. Air bubble entrainment was minimized during packing to ensure a uniform, dense, and continuous column bed. After packing, the upper and lower sieve plates were installed, and the inlet and outlet pipes were connected. The sealing of each interface and the integrity of the flow path were checked.

[0056] Subsequently, ultrapure water was used to vent the entire flow path, and the column bed was then equilibrated with reaction buffer. It was preferred to continuously flush the column bed for about 10 column volumes at a flow rate of 0.6 mL / min to ensure that there were no obvious air bubbles left inside the fixed bed and that the bed reached a stable state, thus completing the basic construction of the fixed bed reactor.

[0057] 1.2 Coupling of T4 phage on the surface of a fixed bed

[0058] After the fixed bed has equilibrated, a 10 mg / mL EDC·HCl solution is introduced into the chromatography column to activate the carboxyl groups on the surface of the carboxyl agarose microspheres. The activation process is preferably carried out in a cyclic manner for 45 min to ensure sufficient contact between the activator and the surface of the microspheres in the fixed bed.

[0059] After activation, drain the activation solution and immediately rinse the column bed with an appropriate amount of reaction buffer to remove unreacted activator and avoid affecting subsequent coupling steps.

[0060] Subsequently, a T4 phage solution was introduced into the activated fixed bed, causing the T4 phage to covalently couple with the activating groups on the surface of the microspheres. The coupling step was preferably performed using a cyclic introduction method to improve the binding efficiency and uniformity of the phage on the fixed bed surface. After coupling, the column bed was thoroughly washed with reaction buffer to remove uncoupled free T4 phage, thus obtaining a phage-microsphere composite fixed bed.

[0061] 1.3 Self-assembly and fixation of CD1.3-Soc enzyme

[0062] After T4 phage conjugation, to reduce non-specific adsorption during subsequent enzyme loading, a blocking buffer containing 2 mg / mL BSA was first introduced into the phage-microsphere composite bed, and the bed was circulated for 6 h. After blocking, the column bed was washed with the appropriate buffer to remove unadsorbed blocking agent.

[0063] The fixed bed was then switched to PI-Mg buffer for equilibration, bringing the system to assembly conditions suitable for Soc tag recognition and binding. After equilibration, CD1.3-Soc enzyme solution was introduced into the column, preferably at a dosage of 2.0 mg, and the column was cycled for 1 h to allow the CD1.3-Soc enzyme to complete directional self-assembly through the specific recognition between the Soc tag and the T4 phage capsid surface site.

[0064] After loading, the column bed is further flushed with PI-Mg buffer or reaction buffer to remove unbound or weakly adsorbed enzyme molecules. Through the above steps, an enzyme-T4 phage-microsphere composite immobilized bed is finally obtained, thereby constructing a continuous flow reactor catalytic platform.

[0065] Specific Example 2: Performance Characterization Method of Continuous Flow Reactor Catalytic Platform

[0066] 2.1 Characterization methods for enzyme loading process

[0067] During the CD1.3-Soc enzyme loading process, samples were collected including the original enzyme solution before loading, the effluent after loading, and the subsequent rinsing solution. SDS-PAGE was used to analyze the protein electrophoresis of these samples to observe changes in the target protein bands, thus determining the loading process of the CD1.3-Soc enzyme in the phage-microsphere composite bed.

[0068] 2.2 Characterization methods for continuous flow catalytic activity

[0069] The enzyme-T4 phage-microsphere composite fixed bed constructed above was retained in a micro medium-pressure chromatography column and connected to a constant flow pump, a sample injection system and an effluent collection device to construct a continuous flow fixed bed catalytic reaction system.

[0070] Using cytidine as a substrate, a substrate solution of appropriate concentration was prepared and continuously introduced into a fixed-bed reactor via a constant-flow pump under set temperature, pH, and flow rate conditions for catalytic reaction. The reaction was initially run for a period to allow the system to reach a stable state, after which effluent samples were collected at set time intervals. The collected effluent samples were terminated by adding methanol, diluted with formic acid aqueous solution, filtered through a filter membrane, and analyzed by high-performance liquid chromatography (HPLC). The catalytic activity of this continuous-flow reactor catalytic platform was characterized by analyzing the changes in substrate and product peak areas.

[0071] 2.3 Characterization methods for comparing the conversion rates of different catalytic systems

[0072] To evaluate the catalytic performance of the enzyme-T4 phage-microsphere composite immobilization system constructed in this invention, it was compared in parallel with the free CD1.3-Soc enzyme system and the CD1.3-Soc enzyme system directly chemically immobilized by microspheres.

[0073] Specifically, using cytidine as a uniform substrate at a concentration of 200 mM, batch reactions were conducted on the free CD1.3-Soc enzyme system, the enzyme-immobilized system directly on the surface of carboxyagarose microspheres, and the enzyme-T4 phage-microsphere composite immobilization system under the same temperature, pH, and reaction time conditions. The batch reaction time for each system was 10 min. After the reaction, reaction samples from each system were collected, and after termination with methanol, dilution with formic acid water, and filtration, substrate consumption and product formation were detected using high-performance liquid chromatography (HPLC), and the substrate conversion rate of each system was calculated. By comparing the conversion rate data of different catalytic systems, the catalytic performance retention effect of the enzyme-T4 phage-microsphere composite immobilization system of this invention compared to the free enzyme system and the directly chemically immobilized enzyme system was characterized.

[0074] 2.4 Characterization method of flow resistance pressure

[0075] We constructed a primitive carboxy agarose microsphere fixed-bed reactor, a phage-microsphere fixed-bed reactor coupled with T4 phage, and an enzyme-T4 phage-microsphere fixed-bed reactor after CD1.3-Soc enzyme self-assembly.

[0076] The three fixed-bed reactor systems were operated under identical equipment and flow rate conditions, and changes in inlet column pressure or system operating pressure were recorded. By comparing pressure data under different fixed-bed reactor conditions, the effects of phage coupling and enzyme self-assembly processes on fluid permeability and bed stability of the fixed-bed reactors were evaluated.

[0077] 2.5 Dynamic Fitting and Characterization Methods for Flow Velocity Response

[0078] To evaluate the kinetic behavior of the continuous flow catalytic platform, different substrate concentration gradients were set up, and continuous flow catalytic reactions were carried out under the same temperature, pH, and flow rate conditions. Effluent samples were collected and analyzed by high-performance liquid chromatography (HPLC) to obtain conversion data under each substrate concentration condition. Based on the obtained experimental data, the apparent kinetic parameters of the continuous flow catalytic platform were determined by fitting the data using a Lilly-Hornby model.

[0079] Meanwhile, to evaluate the impact of flow rate variations on the platform's catalytic performance, different flow rate gradients were set up, and continuous flow catalytic reactions were conducted while maintaining other reaction conditions consistently. Substrate conversion and product formation under different flow rate conditions were measured, and the effects of flow rate on residence time, conversion efficiency, and product output capacity were analyzed.

[0080] 2.6 Characterization Methods for Spatiotemporal Yield

[0081] Under continuous flow catalysis conditions, reactor effluent samples are collected over a fixed time period, and the amount of the target product generated in the samples is determined. Combined with the effective volume of the fixed-bed reactor, the product generation per unit time and per unit reactor volume is calculated, and this is used as the space-time yield of the continuous flow catalysis platform.

[0082] Meanwhile, the batch catalytic system of the free enzyme was tested under the same substrate conditions, and its space-time yield was calculated in the corresponding manner. The process enhancement effect of the continuous flow catalytic platform of the present invention was characterized by comparing the two data.

[0083] 2.7 Characterization methods for cycling stability and storage stability

[0084] The enzyme-T4 phage-microsphere composite material reactor was used for multiple rounds of repeated catalytic experiments. After each round of reaction, the reactor was rinsed with buffer solution, and fresh substrate was introduced under the same conditions for the next round of reaction. Samples were collected after each round of reaction, and the catalytic activity was measured. The activity of each round was compared with the initial activity to characterize the cycle stability of the continuous flow catalytic platform.

[0085] Furthermore, the constructed enzyme-T4 phage-microsphere composite reactor was stored at 4°C in the dark, and its catalytic activity was measured at different storage time points. The substrate was introduced and samples were collected using a continuous flow catalysis method. The formation of the target product was detected by high-performance liquid chromatography (HPLC), and the storage stability of the platform was characterized based on changes in residual activity at different storage times.

[0086] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A continuous flow catalytic system for cytidine deaminase based on a composite material of T4 bacteriophage and agarose microspheres, characterized in that: The system comprises carboxylated agarose microspheres as a fixed-bed substrate, T4 phage as a nanoscaffold, and a CD1.3-Soc fusion enzyme carrying a Soc tag as a functional enzyme. First, T4 phage is covalently coupled to the surface of the carboxylated agarose microspheres via EDC mediation. Then, through the specific recognition between the Soc tag and the binding site on the T4 phage capsid, CD1.3-Soc is directionally self-assembled onto the surface of the T4 phage, yielding an enzyme-T4 phage-microsphere composite catalytic material. This composite catalytic material is loaded into a continuous flow fixed-bed reactor for continuous flow catalytic reactions using cytidine as a substrate.

2. A method for constructing the cytidine deaminase continuous flow catalytic system based on T4 phage and agarose microsphere composite material as described in claim 1, characterized in that: The method is as follows: S1: Carboxylated agarose microspheres are packed into a chromatography column or a continuous flow fixed-bed reactor to form a microsphere fixed-bed substrate; S2: The carboxyl groups on the surface of the carboxylated agarose microspheres are activated by EDC, and T4 phage solution is introduced to covalently couple T4 phage to the surface of the microspheres, resulting in a phage-microsphere composite material; S3: After the phage-microsphere composite material is sealed, CD1.3-Soc enzyme solution is introduced to allow CD1.3-Soc to complete directional self-assembly based on the specific recognition between the Soc tag and the T4 phage capsid surface site, resulting in an enzyme-T4 phage-microsphere composite catalytic material.

3. The construction method according to claim 2, characterized in that: The specific method is as follows: (1) Fixed bed loading: Carboxy agarose microspheres that have been washed with ultrapure water and equilibrated with reaction buffer are loaded into a micro medium-pressure chromatography column by gravity sedimentation to form a 1 mL fixed bed, and the upper and lower sieve plates are installed and the inlet and outlet liquid flow paths are connected. (2) Pretreatment of the flow path: The flow path was vented with ultrapure water and then continuously flushed with reaction buffer at a flow rate of 0.6 mL / min for about 10 column volumes to bring the fixed bed flow path to a stable equilibrium state. (3) Phage coupling: 10 mg / mL EDC·HCl solution was introduced into the packed fixed bed and circulated for 45 min to activate the carboxyl groups on the surface of the carboxyl agarose microspheres; After activation, T4 phage solution was introduced for cyclic coupling, and the column bed was washed with reaction buffer after coupling to remove uncoupled free phage. (4) Blocking treatment: The phage-microsphere composite fixed bed was introduced into the blocking solution containing 2 mg / mL BSA and circulated for 6 h to reduce non-specific adsorption during the subsequent enzyme loading process; after the blocking was completed, the fixed bed was rinsed with buffer. (5) Enzyme self-assembly: The fixed bed was switched to PI-Mg buffer for equilibration, and then 2.0 mg of CD1.3-Soc enzyme solution was injected into the system and circulated for 1 h, so that it could complete directional self-assembly through the specific recognition between the Soc tag and the T4 phage capsid surface site, thereby obtaining the enzyme-T4 phage-microsphere composite fixed bed reactor. (6) Continuous flow catalysis: Cytidine is used as a substrate and is continuously introduced into a fixed bed reactor under set temperature, pH and flow rate conditions to carry out catalytic reaction; after the system reaches steady state, the effluent sample is collected.

4. A method for characterizing the performance of a continuous flow catalytic system for cytidine deaminase based on a composite material of T4 bacteriophage and agarose microspheres, characterized in that, This includes characterizing the system through flow resistance and pressure testing, catalytic activity determination, kinetic fitting, flow rate response analysis, space-time yield comparison, and stability during recycling and storage.

5. The performance characterization method according to claim 5, characterized in that, Catalytic activity determination and space-time yield comparison included: using cytidine as a substrate, the catalytic activity of the enzyme-T4 phage-microsphere composite catalytic material, the directly chemically immobilized enzyme material, and the free CD1.3-Soc enzyme system were determined under the same reaction conditions, and the product generation per unit time per unit reactor volume was compared to evaluate the catalytic efficiency improvement effect of the composite catalytic system under continuous flow conditions.

6. The performance characterization method according to claim 5, characterized in that, The kinetic fitting, flow rate response analysis and flow resistance pressure test include: (1) measuring the conversion data of the continuous flow catalytic system under different substrate concentration conditions, and using the Lilly-Hornby model for kinetic fitting to obtain apparent kinetic parameters; (2) measuring the cytidine conversion rate and corresponding catalytic efficiency under different flow rate conditions, and analyzing the effect of flow rate changes on continuous flow catalytic performance; (3) monitoring the flow resistance pressure changes of the fixed bed reactor under different loading or operating conditions to evaluate the mass transfer performance and bed stability of the composite catalytic material in continuous flow operation.

7. The performance characterization method according to claim 5, characterized in that, The specific tests for recycling stability and storage stability are as follows: (1) The relative activity retention rate of the composite catalytic material during recycling is determined by repeated continuous flow catalysis or multiple rounds of substrate conversion experiments to evaluate its reusability; (2) The composite catalytic material is stored under preset storage conditions, and its remaining catalytic activity is determined at different storage time points to evaluate its storage stability; wherein, the stability evaluation results are used to characterize the operational reliability and application potential of the enzyme-T4 phage-microsphere composite catalytic material in continuous flow biocatalysis applications.

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