Enzyme protein ultrasonic pretreatment and temperature-controlled enzyme acceleration reaction system and operation method

Abstract

The present application relates to the field of enzymatic reaction device, specifically to an enzyme protein ultrasonic pretreatment and temperature control enzyme acceleration reaction system and operation method, which is used to solve the contradiction between enzymatic efficiency and enzyme activity and the problem of insufficient dynamic stability of reaction conditions. The system includes a reaction container, a bearing structure, an ultrasonic transducer array, an ultrasonic generator, a temperature sensor, a temperature control device, an enzymatic reaction monitoring module and a controller; the ultrasonic pretreatment improves the accessibility of the substrate, the temperature control device maintains the stability of the reaction temperature, the monitoring module collects pH and turbidity data in real time, and the controller outputs a termination signal based on the turbidity threshold. The system is mainly used in the enzymatic reaction process of enzyme protein to improve the reaction efficiency and product quality.

Description

Technical Field

[0001] This invention relates to the field of enzymatic reaction apparatus. More specifically, this invention relates to an enzyme-accelerated reaction system and its operating method that involves ultrasonic pretreatment and temperature control of enzyme proteins. Background Technology

[0002] Enzymatic hydrolysis is a key process in bioprocessing and the food industry, and its efficiency and controllability directly affect product yield, quality, and process economy. For a long time, the field has focused on improving the efficiency of enzymatic hydrolysis, typically by optimizing traditional parameters such as reaction temperature, pH, and enzyme-substrate ratio. However, in practice, it has been found that improving the efficiency of enzymatic hydrolysis often faces a fundamental contradiction: excessive pursuit of reaction rate may lead to irreversible loss of enzyme protein activity, while overly conservative reaction conditions result in slow reaction progress and low efficiency.

[0003] Specifically, as a type of biocatalyst, the activity of enzyme proteins is highly dependent on the integrity of their three-dimensional structure. In the initial stages of a reaction, protein substrates often possess a compact higher-order structure, with cleavage sites embedded within the molecule. This makes it difficult for enzyme molecules to effectively contact and act on these sites, resulting in slow reaction initiation and making mass transfer efficiency a limiting step. Although attempts have been made to improve this situation through mechanical stirring, altering the solvent environment, or pretreating the substrate, these methods either have limited effectiveness or may introduce new problems. For example, vigorous mechanical stirring may generate excessive shear forces, directly damaging the fine structure of the enzyme protein; while chemical pretreatment may introduce impurities or alter the chemical environment of the reaction system, making subsequent purification difficult or affecting the quality of the final product.

[0004] Controlling the dynamic stability of enzymatic hydrolysis is also a challenging problem. Enzymatic hydrolysis typically involves the release and absorption of heat, and enzyme activity is extremely sensitive to temperature. Traditional temperature control methods, such as heating or cooling through external jackets, are often limited by their thermal inertia and heat transfer efficiency, making it difficult to achieve rapid and precise control of the internal temperature of the reaction system. Temperature gradients easily form inside the reaction vessel, resulting in localized overheating or underheating zones. Localized overheating may cause denaturation and inactivation of some enzyme proteins, while underheating zones lead to a decrease in the reaction rate. This uneven temperature distribution directly affects the overall efficiency and consistency of the reaction, making it difficult to guarantee the reproducibility of the reaction process.

[0005] Therefore, existing enzymatic hydrolysis reaction systems and methods still face pressing technical challenges in balancing enzymatic hydrolysis efficiency with enzyme activity protection, and in achieving dynamic and stable control of the reaction system (especially temperature). These challenges are interconnected and hinder further improvements in the overall efficiency of enzymatic hydrolysis processes and product quality. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] To achieve these objectives and other advantages according to the present invention, an enzyme-accelerated reaction system for ultrasonic pretreatment and temperature control of enzyme proteins is provided, comprising:

[0008] The reaction vessel has an internal support structure for holding enzymes and protein substrates, and an array of ultrasonic transducers is installed at the bottom of the reaction vessel.

[0009] An ultrasonic generator, whose output is connected to an ultrasonic transducer array via wires, is configured to generate a frequency of 20-40kHz and a power density of 0.5-2W / cm². 2 The ultrasonic signal;

[0010] A temperature sensor is embedded in the side wall of the reaction vessel, and the probe of the temperature sensor is placed in the internal solution layer of the reaction vessel.

[0011] A temperature control device, whose input end is connected to the signal output end of a temperature sensor, includes a heating element and a cooling circulation pipeline. The heating element is attached to the outer wall surface of the reaction vessel, and the cooling circulation pipeline surrounds the outer wall of the reaction vessel and is connected to an external refrigerator.

[0012] The enzymatic hydrolysis reaction monitoring module includes a pH detection unit and a real-time turbidity sensor. The electrodes of the pH detection unit and the probe of the real-time turbidity sensor are both immersed in the internal solution layer of the reaction vessel. The enzymatic hydrolysis reaction monitoring module is configured to collect the pH value and turbidity data of the solution in real time.

[0013] The controller is connected to the ultrasonic generator, the temperature control device, and the enzymatic reaction monitoring module. The controller is configured to control the ultrasonic generator to run at a preset frequency for a preset time and then turn it off; after the ultrasonic generator is turned off, the controller controls the temperature control device to adjust and maintain the temperature in the reaction vessel at 30-60℃; the controller receives turbidity data collected by the enzymatic reaction monitoring module and outputs a termination signal to terminate the reaction when the turbidity data reaches a preset threshold.

[0014] Preferably, the controller is further configured to receive pH data collected by the pH detection unit and calculate the rate of change of the pH data; when the turbidity data reaches a preset threshold, it verifies whether the rate of change of the pH data is within the preset rate of change range; and only when the turbidity data reaches the preset threshold and the rate of change of the pH data is within the preset rate of change range, it outputs a termination signal to terminate the reaction.

[0015] Preferably, the controller is further configured to calculate the rate of change of turbidity data over time in real time, i.e., the turbidity change rate, and only output a termination signal to terminate the reaction when the following three conditions are met simultaneously:

[0016] Condition a: Turbidity data reaches a preset threshold;

[0017] Condition b: The rate of change of pH data is within its preset range.

[0018] Condition c: The rate of change of turbidity is lower than a preset positive threshold.

[0019] Preferably, the controller is also configured to: when the controller outputs a termination signal, the controller controls the heating element of the temperature control device to raise the temperature inside the reaction vessel to above 80°C within 1 minute and maintain it for at least 2 minutes.

[0020] Preferably, the controller has at least one ultrasound parameter combination database pre-stored, which associates different types of enzyme proteins with preferred combinations of ultrasound frequency, power density and processing time.

[0021] The controller is configured to receive current enzyme protein type information input by the user or identified by sensors, and to retrieve the corresponding ultrasonic frequency, power density and processing time from the ultrasonic parameter combination database based on the current enzyme protein type information in order to control the operation of the ultrasonic generator.

[0022] Preferably, it also includes a stirring mechanism, which includes:

[0023] A drive motor, which is fixed to the top or bottom of the reaction vessel;

[0024] The stirring shaft has one end connected to the output end of the drive motor and the other end extending to the internal solution layer of the reaction vessel. The supporting structure is fixed on the stirring shaft and is a mesh basket.

[0025] The drive motor is electrically connected to the controller, which is further configured to control the drive motor to operate in a first speed range during the operation of the ultrasonic generator; and to control the drive motor to operate in a second speed range, which is lower than the first speed range, during the enzymatic reaction stage after the ultrasonic generator is turned off.

[0026] Preferably, the mesh basket is composed of two layers of mesh walls, forming a hollow sandwich structure. The mesh diameter of the inner layer is 50-200μm, and the mesh diameter of the outer layer is 1-5mm. Its mechanical strength is higher than that of the inner layer. The side walls or top cover of the mesh basket are provided with openable and closable filling ports.

[0027] Preferably, a sampling tube with a valve is provided at the bottom of the side wall of the reaction vessel.

[0028] A method for operating an enzyme-accelerated reaction system involving ultrasonic pretreatment and temperature control of enzyme proteins is provided, comprising the following steps:

[0029] S1. Place the enzyme and protein substrate in the support structure of the reaction vessel, and add buffer solution into the reaction vessel until the support structure is completely submerged;

[0030] S2. Start the ultrasonic generator through the controller, so that the ultrasonic transducer array runs for a preset time according to the preset parameters to perform ultrasonic pretreatment on the enzymes and protein substrates in the carrier structure.

[0031] S3. After turning off the ultrasonic generator, use the controller to control the temperature control device to adjust and maintain the temperature inside the reaction vessel within the set temperature range required for the enzymatic hydrolysis reaction.

[0032] S4. The pH value and turbidity data of the solution are collected in real time through the enzymatic hydrolysis reaction monitoring module and transmitted to the controller;

[0033] S5. The controller outputs a termination signal to terminate the reaction when the turbidity data reaches a preset threshold, based on the received turbidity data.

[0034] The present invention has at least the following beneficial effects:

[0035] First, this invention systematically integrates ultrasonic pretreatment with specific parameters, precise temperature control, and turbidity-based endpoint determination. The ultrasonic pretreatment step effectively improves the structural accessibility of protein substrates at the physical level, creating more favorable initial conditions for subsequent enzymatic hydrolysis. The integrated temperature control device overcomes the thermal inertia problem of traditional temperature control methods through rapid heating and cooling responses, significantly improving the thermal stability of the reaction system. This multi-module synergy, working together from reaction initiation and process maintenance to endpoint determination, effectively alleviates the inherent contradiction between enzymatic hydrolysis efficiency and enzyme activity protection, improving the controllability and efficiency of the entire reaction process.

[0036] Secondly, this invention introduces pH change rate as a second judgment parameter, forming a dual-parameter verification mechanism with turbidity data, which significantly improves the intelligence and reliability of reaction endpoint determination. It can effectively distinguish between turbidity changes caused by the actual reaction process and interference signals caused by bubbles, non-reactive particles, or sensor noise. This cross-verification based on the essential characteristics of the chemical reaction (pH change) and physical phenomena (turbidity change) reduces reliance on a single sensor, avoids premature or delayed termination due to misjudgment, and thus more accurately captures the reaction endpoint in complex reaction environments, further optimizing the quality and consistency of reaction products.

[0037] Third, based on dual-parameter verification, this invention further introduces the dynamic criterion of turbidity change rate, constructing a triple verification logic combining static threshold and dynamic trend. This can keenly capture the essential characteristic of a significant decrease in kinetic rate as the reaction approaches its endpoint, thus elevating endpoint judgment from relying on instantaneous values ​​to a higher level of analyzing process trends. This intelligent decision-making algorithm based on multi-parameter temporal correlation features effectively filters instantaneous interference without increasing hardware costs, greatly enhancing the robustness and accuracy of the system in determining the endpoint in complex environments.

[0038] Fourth, this invention establishes a database of ultrasound parameter combinations associated with enzyme protein types, enabling the system to perform personalized pretreatment for different enzyme protein characteristics. This overcomes the limitations of a one-size-fits-all approach using fixed ultrasound parameters, allowing for customized pretreatment schemes for enzyme proteins with varying structural sensitivities. This intelligent matching mechanism enables ultrasound pretreatment to optimize the pretreatment effect on specific enzyme-substrate systems while minimizing damage to enzyme activity, significantly improving the system's applicability, reproducibility of treatment effects, and level of intelligence.

[0039] Fifth, this invention balances the conflicting technical requirements under the synergistic effect of ultrasound and stirring by employing a specific double-layer sandwich structure design for the mesh basket. The inner small-pore mesh wall ensures effective retention of enzymes and substrates, while the outer large-pore high-strength mesh wall protects against ultrasonic cavitation impacts and fluid shear forces, while ensuring smooth solution flow. This functionally partitioned structural design improves the durability and reliability of the device, optimizes mass transfer efficiency, and also ensures ease of operation, providing crucial hardware support for the stable and efficient operation of the entire reaction system.

[0040] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the connection of an enzyme-accelerated reaction system according to one of the technical solutions of the present invention.

[0042] Explanation of reference numerals in the accompanying drawings: 1. Reaction vessel; 2. Support structure; 3. Ultrasonic transducer array; 4. Ultrasonic generator; 5. Temperature sensor; 6. Temperature control device; 7. Controller; 8. pH detection unit; 9. Turbidity sensor; 10. Drive motor; 11. Stirring shaft; 12. Filling port; 13. Top cover; 14. External refrigeration unit. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0044] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0045] like Figure 1 As shown, this embodiment provides an enzyme-accelerated reaction system for ultrasonic pretreatment and temperature control of enzyme proteins, which mainly includes: a reaction container 1, a support structure 2, an ultrasonic transducer array 3, an ultrasonic generator 4, a temperature sensor 5, a temperature control device 6, an enzyme hydrolysis reaction monitoring module, and a controller 7.

[0046] The reaction vessel is a cylindrical container with a sealed lid made of 316L stainless steel, used to hold the buffer solution. The interior of the reaction vessel has a support structure; in this embodiment, the support structure is a mesh basket made of 316L stainless steel, used to hold the enzyme and protein substrate. The ultrasonic transducer array is fixedly mounted on the bottom outer wall of the reaction vessel using waterproof sealant.

[0047] The ultrasonic generator is placed on the workbench next to the reaction vessel, and its output is connected to the ultrasonic transducer array via shielded wires. The ultrasonic generator is configured to produce an adjustable frequency in the range of 20-40kHz and a power density of 0.5-2W / cm². 2 Adjustable ultrasonic signals within a certain range.

[0048] The temperature sensor is a Pt100 platinum resistance temperature sensor, whose sensing probe is embedded in the side wall of the reaction vessel and extends into the internal solution layer of reaction vessel 1 for direct measurement of solution temperature.

[0049] The temperature control device includes a silicone heating film (i.e., a heating element) attached to the outer wall of the reaction vessel, and copper tubing (i.e., a cooling circulation system) coiled around the outer wall of the reaction vessel. The cooling circulation system is connected to an external circulating water chiller (i.e., an external chiller 14) via an insulated pipe. The signal output terminal of the temperature sensor is connected to the input terminal of the temperature control device.

[0050] The enzymatic hydrolysis reaction monitoring module includes a composite pH electrode (i.e., pH detection unit 8) and an insertion-type turbidity sensor 9 (i.e., real-time turbidity sensor). Both the electrode of the pH detection unit and the probe of the real-time turbidity sensor are immersed in the internal solution layer of the reaction vessel through the opening in the top cover 13. The enzymatic hydrolysis reaction monitoring module is configured to acquire pH and turbidity NTU data of the solution once per second.

[0051] The controller is an embedded industrial computer that establishes communication connections with the ultrasonic generator, temperature control device, and enzymatic reaction monitoring module via data cables.

[0052] The effect of ultrasonic pretreatment on enzymatic hydrolysis: The inventors believe that ultrasonic pretreatment with specific parameters (frequency 20-40kHz, power density 0.5-2.0W / cm³) is effective. 2 (Time 5-15 min) can significantly improve the efficiency of subsequent enzymatic hydrolysis reactions. Its mechanism of action is mainly based on the following aspects:

[0053] The cavitation effect generated when ultrasound propagates in a liquid medium is its main mechanism of action. This effect can generate extremely high instantaneous pressure and temperature locally, producing strong mechanical shear forces on protein substrates. This action can effectively disrupt the compact higher-order structure of protein substrates, causing them to partially denature and extend, exposing more enzyme cleavage sites that were originally embedded inside the molecule, thereby significantly increasing the contact area between the enzyme and the substrate and the probability of reaction.

[0054] The microjets and intense vibrations generated by ultrasound in solution can significantly enhance the mass transfer process within the reaction system. This not only promotes the diffusion and collision frequency of enzyme and substrate molecules, but also helps to overcome the liquid film resistance at the reaction interface, making the enzymatic reaction more rapid and uniform.

[0055] Within a mild parameter range, ultrasound may cause a certain degree of perturbation to the enzyme protein molecules themselves, inducing a moderate change in their conformation that is conducive to the catalytic reaction, thereby activating the enzyme molecules to a certain extent and improving their instantaneous catalytic efficiency, while avoiding the risk of irreversible enzyme inactivation that may be caused by high-intensity ultrasound.

[0056] Therefore, through the synergistic effect of the aforementioned multiple physical effects, the ultrasonic pretreatment step creates more favorable initial conditions for the subsequent enzymatic hydrolysis reaction, resulting in faster reaction initiation and deeper progress. This manifests macroscopically as a significant reduction in enzymatic hydrolysis reaction time and an effective increase in the yield of the final product. The parameter range selected in this invention represents an optimized balance between fully activating the aforementioned beneficial effects and avoiding damage to enzyme activity.

[0057] Experimental group: The system using this implementation method is operated as follows:

[0058] Trypsin and casein substrate were placed in a mesh basket, and phosphate buffer solution (pH=7.8) was added to the reaction vessel until the support structure was completely submerged. The ultrasonic pretreatment parameters were set via the controller to: frequency 25kHz, power density 1.0W / cm³. 2 The processing time is 10 minutes. After pretreatment, the system automatically shuts down the ultrasonic generator and controls the reaction system temperature at 50℃ for enzymatic hydrolysis. The turbidity is monitored in real time by the enzymatic hydrolysis reaction monitoring module. When the turbidity value reaches 1500 NTU, the controller outputs a termination signal, and the system performs thermal inactivation to terminate the reaction.

[0059] Control group: Except for the absence of ultrasonic pretreatment, all other conditions (including the type and amount of enzyme and substrate, buffer solution, reaction temperature of 50℃, termination conditions, etc.) were exactly the same as those of the experimental group. The enzyme and substrate were placed directly in a 50℃ environment to start the enzymatic hydrolysis reaction.

[0060] The product yield and enzymatic hydrolysis rate of the two groups of experiments were measured and compared: the product yield of the experimental group (pretreated with ultrasound) was 15.2% higher than that of the control group. The time required for the experimental group to reach the termination condition (turbidity 1500 NTU) from the start of the enzymatic hydrolysis reaction was 45 min, while the time required for the control group to reach the same termination condition was 72 min. The enzymatic hydrolysis rate of the experimental group was significantly higher than that of the control group. The results show that the system of the present invention, by introducing an ultrasound pretreatment step with specific parameters, can effectively improve the efficiency of the enzymatic hydrolysis reaction, specifically by significantly shortening the reaction time and increasing the final product yield.

[0061] In another embodiment of the present invention, a method for determining the endpoint of an enzymatic hydrolysis reaction is provided. Based on real-time monitoring of turbidity data, a pH change rate is further introduced as a co-criterion, constructing a dual-parameter verification mechanism. This aims to solve the technical problem that a single turbidity criterion is easily interfered with, leading to misjudgment of the endpoint. This is illustrated in the following example:

[0062] Example 1: A method for determining the endpoint of an enzymatic hydrolysis reaction based on dual-parameter validation, employing a dual-parameter collaborative validation mechanism using turbidity data and pH change rate, including:

[0063] The reaction vessel contains a mesh basket for carrying enzymes and protein substrates. An array of ultrasonic transducers is located at the bottom of the reaction vessel.

[0064] The enzymatic hydrolysis reaction monitoring module includes:

[0065] The pH detection unit uses a composite pH electrode; the real-time turbidity sensor uses an insertion-type turbidity probe; and the controller uses an embedded industrial computer.

[0066] Implementation steps:

[0067] (1) Place the trypsin and casein substrate in a mesh basket and add phosphate buffer solution until completely submerged;

[0068] (2) Start ultrasonic pretreatment: frequency 28kHz, power density 1.2W / cm² 2 Processing time: 8 minutes;

[0069] (3) Turn off the ultrasound and maintain the temperature of the reaction system at 37°C;

[0070] (4) The controller executes the following judgment logic:

[0071] Real-time data collection: Turbidity (T (NTU)) and pH value;

[0072] Real-time calculation: pH change rate R = ΔpH / Δt (pH / min);

[0073] Judgment condition: If T≥T set And R min ≤R≤R max If the condition is met, a termination signal is output; otherwise, the response continues.

[0074] Wherein: Turbidity preset threshold T set =1800 NTU; pH change rate preset range R min =-0.015pH / min, R max =0.015pH / min; sampling interval Δt=1s; pH change rate was calculated using the sliding window method with a window width of 10 sampling points.

[0075] The experimental group (Example 1) used the two-parameter verification method of this embodiment, while the control group only used turbidity as a single parameter. The results showed that the experimental group had accurate reaction termination timing, increased product yield by 12.3%, and reduced enzyme activity loss by 15.6%. The control group experienced two misjudgments due to bubble interference, resulting in significant fluctuations in product yield.

[0076] Example 2: Parameter optimization for acidic proteases. Building upon Example 1, parameter optimization is performed specifically for the characteristics of acidic proteases:

[0077] Reaction temperature: 45℃; Turbidity preset threshold: 2200 NTU; pH change rate preset range: -0.008 pH / min to 0.008 pH / min; Buffer system: Citrate-disodium hydrogen phosphate buffer.

[0078] This invention uses the pH change rate as a second judgment parameter to complement turbidity data for verification, effectively distinguishing the true reaction endpoint from sensor interference signals. This dual-parameter verification mechanism significantly improves the accuracy and reliability of reaction endpoint determination and solves the technical problem of single turbidity sensors being susceptible to interference.

[0079] In another embodiment of the present invention, a method for determining the endpoint of an enzymatic hydrolysis reaction is provided. Based on dual-parameter verification, the turbidity change rate is further introduced as a third criterion, constructing a triple verification mechanism based on a combination of static threshold and dynamic trend. This aims to more accurately identify the reaction kinetic endpoint and improve the robustness of judgment under complex interference environments. The following example illustrates this further:

[0080] Example 3: Based on the three-parameter endpoint determination of turbidity threshold, pH change rate, and turbidity change rate, and building upon Example 1 or 2, a triple verification mechanism is constructed by introducing the turbidity change rate as a third determination parameter. In the configuration, the controller is further configured to execute the following processing logic:

[0081] Real-time acquisition of turbidity and pH data;

[0082] Real-time calculation of pH change rate R pH =ΔpH / Δt;

[0083] Real-time calculation of turbidity change rate R Turb =ΔT urb / Δt;

[0084] The following three conditions are used for collaborative judgment.

[0085] The judgment conditions include:

[0086] Condition a: Turbidity data ≥ preset turbidity threshold T set ;

[0087] Condition b: pH change rate R pH Within the preset range [R] pHmin R pHmax ]Inside;

[0088] Condition c: Turbidity change rate R Turb ≤Preset positive threshold R Turbmax .

[0089] The controller outputs a termination signal to terminate the reaction only when conditions a, b, and c are all met simultaneously.

[0090] In one specific implementation: a preset turbidity threshold T set =2000 NTU; pH change rate range set to [-0.012, +0.012] pH / min; turbidity change rate threshold R Turbmax =5 NTU / min; sampling interval Δt=1s; the rate of change was calculated using the moving average method, and the window width was 10 sampling points.

[0091] By introducing the dynamic parameter of turbidity change rate, the kinetic characteristics of a reaction approaching its endpoint can be effectively identified. When the reaction substrate is essentially exhausted, the turbidity change rate will significantly decrease and tend to stabilize. This parameter can effectively distinguish the true reaction endpoint from instantaneous turbidity fluctuations caused by bubble, particle aggregation, or sensor noise, thus providing a more reliable endpoint determination in complex reaction environments.

[0092] This invention is not limited to the specific parameter values ​​mentioned above. Those skilled in the art can adjust the above thresholds and ranges according to the characteristics of different enzyme-substrate systems, and these adjustments still fall within the protection scope of this invention.

[0093] In another embodiment of the present invention, the system further includes a termination execution module electrically connected to the controller. This termination execution module is not a separate hardware device, but rather its function is implemented by the controller calling the system's existing temperature control device through programming logic. Specifically, when the controller outputs a termination signal based on preset judgment logic (such as based on turbidity data or a multi-parameter verification mechanism), this signal will trigger the controller to execute a predetermined termination procedure.

[0094] During the enzymatic hydrolysis reaction, when the controller determines that the reaction has reached its endpoint and outputs a termination signal, the controller immediately controls the heating element of the temperature control device (such as the previously mentioned silicone heating film) to enter a high-intensity operating mode. Specifically, the controller controls the heating element to rapidly raise the solution temperature in the reaction vessel from the enzymatic hydrolysis reaction temperature (e.g., 30-60°C) to 85°C within 1 minute, and maintains this temperature for 3 minutes. After this high-temperature maintenance phase, the system can automatically stop heating, or the controller can activate the cooling circulation pipeline to cool down, thereby completing the termination operation of the entire enzymatic hydrolysis reaction.

[0095] This embodiment utilizes the system's inherent temperature control device as a termination module and sets its specific operating mode to achieve rapid, thorough, and physical termination of the enzymatic hydrolysis reaction without the introduction of external chemical substances. This approach ensures that the reaction can be terminated promptly and effectively at the optimal moment determined by the controller, avoiding over-enzymatic hydrolysis or degradation of the target product due to termination delays, and also eliminating product contamination problems that may arise from the addition of chemical terminators.

[0096] In another embodiment of the present invention, the controller pre-stores at least one database of ultrasonic parameter combinations. This database is stored in the form of a data table, which records the association between different types of enzyme proteins and their preferred combinations of ultrasonic frequency, power density, and processing time.

[0097] Before the system initiates the ultrasonic pretreatment procedure, the controller is configured to receive the current enzyme protein type information (e.g., selection of "trypsin" or "acidic protease") input by the user through a human-machine interface. Alternatively, in another optional implementation, the system may be equipped with a simple biosensor to automatically identify and feed back the current enzyme protein type information to the controller.

[0098] After acquiring the current enzyme protein type information, the controller queries the ultrasound parameter combination database based on this information and retrieves the corresponding preferred ultrasound frequency, power density, and processing time. For example, when the current enzyme protein type is "trypsin," the controller retrieves and sets the ultrasound frequency to 28kHz and the power density to 1.2W / cm² from the database. 2 The processing time is 8 minutes. The controller controls the operation of the ultrasonic generator based on the parameters called in this group to complete the targeted ultrasonic pretreatment steps.

[0099] This implementation introduces a database of ultrasound parameter combinations associated with enzyme protein types, enabling the system to tailor pretreatment schemes for different enzyme proteins. This solves the technical challenge that a fixed range of ultrasound parameters may not be suitable for the diverse characteristics of enzyme proteins, achieving personalization and intelligence in the pretreatment stage. By maximizing the pretreatment efficiency of ultrasound on specific enzyme-substrate systems while avoiding damage to enzyme activity, the final enzymatic reaction effect is optimized and stabilized at a deeper level, improving the system's applicability and the reproducibility of the treatment effect.

[0100] In another embodiment of the present invention, the system further includes a stirring mechanism. The stirring mechanism specifically includes:

[0101] The drive motor 10 is fixed to the top of the reaction vessel by a bracket;

[0102] A stirring shaft 11 has one end connected to the output end of a drive motor via a coupling, and the other end extending into the internal solution layer of the reaction vessel; and a support structure, which is a mesh basket, is disposed on the stirring shaft. The drive motor is electrically connected to a controller and receives speed commands from the controller.

[0103] The controller is further configured to control the drive motor to operate within a first speed range during the operation of the ultrasonic generator (i.e., the ultrasonic pretreatment stage). This first speed range is set to 200-400 r / min, the purpose of which is to create a gentle turbulence in the solution, using the macroscopic flow of mechanical stirring to disperse and homogenize the instantaneous hot spots or local high-temperature areas generated by the ultrasonic cavitation effect, thereby helping to avoid potential damage to the enzyme protein from local overheating at the system level.

[0104] During the enzymatic hydrolysis stage after the ultrasonic generator is turned off, the controller directs the drive motor to operate within a second speed range. This second speed range is set to 50-150 r / min, which is lower than the first speed range. This reduced stirring speed aims to maintain the overall homogeneity of the reaction solution, ensuring sufficient contact between the enzyme and substrate to maintain reaction efficiency, while avoiding unnecessary damage to the enzyme molecular structure caused by excessive mechanical shear force generated by high-speed stirring.

[0105] This embodiment solves the technical challenge of simultaneously eliminating localized ultrasonic thermal effects and avoiding enzyme shear damage through a single stirring mode by employing a precisely controlled stirring mechanism with differentiated stirring strategies for the two different stages of ultrasonic pretreatment and enzymatic hydrolysis. This staged stirring logic, working in conjunction with the ultrasonic-temperature control system, further enhances the uniformity and stability of the reaction system, providing a favorable physical environment for the efficient and stable enzymatic hydrolysis reaction.

[0106] In another embodiment of the present invention, the supporting structure in the stirring mechanism, namely the mesh basket, has a specific double-layer sandwich configuration. Specifically, the mesh basket consists of inner and outer mesh walls, forming a hollow sandwich structure for containing enzymes and protein substrates. The inner mesh wall has a mesh diameter of 50-200 μm, and its function is to trap enzymes and small protein substrates, preventing them from leaking into the bulk solution of the reaction vessel. The outer mesh wall has a mesh diameter of 1-5 mm, and its mechanical strength is higher than that of the inner mesh wall. The outer mesh wall directly withstands the severe impact generated by the ultrasonic cavitation effect and the shear force generated by the solution flow, thereby providing mechanical protection for the inner mesh wall. A closable filling port 12 is provided on the side wall of the mesh basket for loading or unloading enzymes and protein substrates, facilitating operation.

[0107] This embodiment achieves precise functional zoning through a specific structural design of the mesh basket. The inner small-pore mesh wall ensures effective retention and fixation of the enzyme and substrate, while the outer large-pore high-strength mesh wall plays a major role in protecting against mechanical stress, significantly improving the durability and reliability of the mesh basket under the combined effects of ultrasound and stirring. Simultaneously, the large-pore design of the outer layer provides unobstructed flow channels for the reaction solution, minimizing the mesh basket's obstruction of the mass transfer process and ensuring efficient mass transfer during the ultrasonic pretreatment and enzymatic hydrolysis stages. The openable / closable filling port structure enhances operational convenience. This optimized design for a specific application scenario balances multiple requirements such as retention, mass transfer, shock resistance, and ease of operation, further enhancing the overall performance and stability of the entire reaction system.

[0108] In another embodiment of the invention, the reaction vessel is a cylindrical container with a sealed cap made of 316L stainless steel, used to hold a buffer solution. A sampling port is provided at the bottom of the side wall of the reaction vessel, and a sampling tube with a valve is installed thereon. The sampling tube is made of corrosion-resistant 316L stainless steel, with an inner diameter of 3 mm, an outer diameter of 5 mm, and a length extending approximately 100 mm beyond the reaction vessel. The sampling tube is sealed to the reaction vessel wall by welding, and a manual stopcock valve is installed at its outer end. The valve is made of polytetrafluoroethylene (PTFE) to ensure good sealing and chemical inertness. The valve is configured to completely seal the sampling tube when closed, preventing leakage of the reaction solution; when sampling is required, the valve is opened by manually rotating it, allowing a small amount of reaction solution to flow from inside the reaction vessel to an external receiving container.

[0109] The valved sampling tube allows operators to conveniently and non-destructively extract small samples of the reaction solution during the reaction process (such as after ultrasonic pretreatment or during enzymatic hydrolysis) without opening the sealed lid of the reaction vessel. To sample, the operator simply places the external receiving container below the sampling tube outlet, slowly opens the valve, and allows the hydrostatic pressure within the reaction vessel to allow the reaction solution to flow out naturally. After collecting approximately 1 to 5 mL of sample, the valve is closed. The collected samples can be used for immediate offline analysis, such as verifying the accuracy of the system's built-in sensors, detecting intermediate products, or conducting parallel experimental studies. The sampling tube's installation position at the bottom of the side wall ensures that the sample represents the overall composition of the solution within the reaction vessel, avoiding the influence of sediment or air bubbles.

[0110] In another embodiment of the present invention, a method for operating an enzyme-accelerated reaction system for ultrasonic pretreatment and temperature control of enzyme proteins is provided, comprising the following steps:

[0111] S1. Place the enzyme and protein substrate in the support structure of the reaction vessel, and add buffer solution into the reaction vessel until the support structure is completely submerged;

[0112] S2. The ultrasonic generator is started via the controller, causing the ultrasonic transducer array to run for a preset time according to preset parameters to perform ultrasonic pretreatment on the enzymes and protein substrates in the support structure; wherein the preset parameters include ultrasonic frequency 20-40kHz and power density 0.5-2W / cm³. 2 The preset time is 5-15 minutes;

[0113] S3. After turning off the ultrasonic generator, use the controller to control the temperature control device to adjust and maintain the temperature inside the reaction vessel within the enzymatic hydrolysis reaction set temperature range of 30-60℃.

[0114] S4. The pH value and turbidity data of the solution are collected in real time through the enzymatic hydrolysis reaction monitoring module and transmitted to the controller;

[0115] S5. The controller outputs a termination signal to terminate the reaction when the turbidity data reaches a preset threshold, based on the received turbidity data.

[0116] The operating method of this invention integrates ultrasonic pretreatment, isothermal enzymatic hydrolysis, and endpoint determination based on real-time monitoring into a coherent and automated process flow. The startup sequence, working logic, and collaborative relationships of each functional module are clearly defined, allowing the innovative functions of the aforementioned hardware devices to be systematically utilized. Through this procedural operating process, the complex control of the enzymatic hydrolysis reaction is transformed into repeatable and standardized steps, significantly reducing operational difficulty and human intervention, ensuring the consistency of the reaction process and the reproducibility of results, and providing clear methodological guidance for achieving efficient and stable enzymatic hydrolysis reactions.

[0117] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. An enzyme-accelerated reaction system for ultrasonic pretreatment and temperature control of enzyme proteins, characterized in that, include: The reaction vessel has an internal support structure for holding enzymes and protein substrates, and an array of ultrasonic transducers is installed at the bottom of the reaction vessel. An ultrasonic generator, whose output is connected to an ultrasonic transducer array via wires, is configured to generate a frequency of 20-40kHz and a power density of 0.5-2W / cm². 2 The ultrasonic signal; A temperature sensor is embedded in the side wall of the reaction vessel, and the probe of the temperature sensor is placed in the internal solution layer of the reaction vessel. A temperature control device, whose input end is connected to the signal output end of a temperature sensor, includes a heating element and a cooling circulation pipeline. The heating element is attached to the outer wall surface of the reaction vessel, and the cooling circulation pipeline surrounds the outer wall of the reaction vessel and is connected to an external refrigerator. The enzymatic hydrolysis reaction monitoring module includes a pH detection unit and a real-time turbidity sensor. The electrodes of the pH detection unit and the probe of the real-time turbidity sensor are both immersed in the internal solution layer of the reaction vessel. The enzymatic hydrolysis reaction monitoring module is configured to collect the pH value and turbidity data of the solution in real time. The controller is connected to the ultrasonic generator, the temperature control device, and the enzymatic hydrolysis reaction monitoring module. The controller is configured to control the ultrasonic generator to run at a preset frequency for a preset time and then turn it off; after the ultrasonic generator is turned off, the controller controls the temperature control device to adjust and maintain the temperature in the reaction vessel at 30-60℃; and receives turbidity data collected by the enzymatic hydrolysis reaction monitoring module. The controller is configured to receive pH data collected by the pH detection unit and calculate the rate of change of the pH data; The controller is configured to calculate the rate of change of turbidity data over time in real time, i.e., the turbidity change rate, and only output a termination signal to stop the reaction when the following three conditions are met simultaneously: Condition a: Turbidity data reaches a preset threshold; Condition b: The rate of change of pH data is within its preset range. Condition c: The rate of change of turbidity is lower than a preset positive threshold.

2. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 1, characterized in that, The controller is also configured to: when the controller outputs a termination signal, the controller controls the heating element of the temperature control device to raise the temperature inside the reaction vessel to above 80°C within 1 minute and maintain it for at least 2 minutes.

3. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 1, characterized in that, The controller has at least one ultrasound parameter combination database pre-stored, which associates different types of enzyme proteins with combinations of ultrasound frequency, power density and processing time. The controller is configured to receive current enzyme protein type information input by the user or identified by sensors, and to retrieve the corresponding ultrasonic frequency, power density and processing time from the ultrasonic parameter combination database based on the current enzyme protein type information in order to control the operation of the ultrasonic generator.

4. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 1, characterized in that, It also includes a stirring mechanism, which includes: A drive motor, which is fixed to the top or bottom of the reaction vessel; The stirring shaft has one end connected to the output end of the drive motor and the other end extending to the internal solution layer of the reaction vessel. The supporting structure is fixed on the stirring shaft and is a mesh basket. The drive motor is electrically connected to the controller, which is configured to control the drive motor to operate in a first speed range during the operation of the ultrasonic generator; and to control the drive motor to operate in a second speed range, which is lower than the first speed range, during the enzymatic reaction stage after the ultrasonic generator is turned off.

5. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 4, characterized in that, The mesh basket is composed of two layers of mesh walls, forming a hollow sandwich structure. The mesh diameter of the inner layer is 50-200μm, and the mesh diameter of the outer layer is 1-5mm. Its mechanical strength is higher than that of the inner layer. The side walls or top cover of the mesh basket are provided with openable and closable filling ports.

6. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 1, characterized in that, A sampling tube with a valve is installed at the bottom of the side wall of the reaction vessel.

7. A method for operating an enzyme-accelerated reaction system for ultrasonic pretreatment and temperature control of enzyme proteins as described in any one of claims 1 to 6, comprising the following steps: S1. Place the enzyme and protein substrate in the support structure of the reaction vessel, and add buffer solution into the reaction vessel until the support structure is completely submerged; S2. Start the ultrasonic generator through the controller, so that the ultrasonic transducer array runs for a preset time according to the preset parameters to perform ultrasonic pretreatment on the enzymes and protein substrates in the carrier structure. S3. After turning off the ultrasonic generator, use the controller to control the temperature control device to adjust and maintain the temperature inside the reaction vessel within the set temperature range required for the enzymatic hydrolysis reaction. S4. The pH value and turbidity data of the solution are collected in real time through the enzymatic hydrolysis reaction monitoring module and transmitted to the controller; S5. The controller outputs a termination signal to terminate the reaction based on the received turbidity data and when the turbidity data reaches a preset threshold, the rate of change of pH data is within its preset rate of change range, and the rate of change of turbidity is lower than a preset positive threshold.

Images