Enzyme accelerated reaction system for enzyme protein ultrasonic pretreatment and temperature control and operation method

Abstract

The invention relates to the field of enzymolysis reaction devices, in particular to an enzyme accelerated reaction system for ultrasonic pretreatment and temperature control of zymoprotein and an operation method, which are used for solving the problems of contradiction between enzymolysis efficiency and enzyme activity and insufficient dynamic stability of reaction conditions. The system comprises a reaction container, a bearing structure, an ultrasonic transducer array, an ultrasonic generator, a temperature sensor, a temperature control device, an enzymolysis reaction monitoring module and a controller, the substrate accessibility is improved through ultrasonic pretreatment, the temperature control device keeps the reaction temperature stable, the monitoring module collects pH and turbidity data in real time, and the controller outputs a termination signal based on a turbidity threshold value. The system is mainly used for the enzymolysis reaction process of zymoprotein so as to improve the reaction efficiency and the product quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzymatic reaction device. More particularly, the present application relates to an ultrasonic pretreatment and temperature-controlled enzyme acceleration reaction system and operation method. BACKGROUND

[0002] As a key process in biological processing and food industry, the efficiency and controllability of enzymatic reaction directly affect the product yield, quality and process economy. For a long time, the field has been committed to improving the efficiency of enzymatic reaction, usually focusing on optimizing reaction temperature, pH value, enzyme and substrate ratio and other traditional parameters. However, in practice, it is found that the efficiency improvement of enzymatic reaction often faces a fundamental contradiction: that is, excessive pursuit of reaction rate may lead to irreversible loss of enzyme activity, and too conservative reaction conditions make the reaction process slow and inefficient.

[0003] Specifically, as a biological catalyst, the activity of enzyme protein is highly dependent on the integrity of its three-dimensional structure. In the early stage of reaction, the protein substrate often has a tight higher structure, and the enzyme cleavage site is embedded in the interior of the molecule, making it difficult for the enzyme molecule to effectively contact and act on these sites, resulting in slow reaction start and mass transfer efficiency as the limiting step. Although the field attempts to improve this situation by mechanical stirring, changing the solvent environment or pretreating the substrate, these methods either have limited effect or may introduce new problems. For example, strong mechanical stirring may generate excessive shear force, directly damaging the delicate structure of enzyme protein; and chemical pretreatment may introduce impurities or change the chemical environment of the reaction system, making subsequent purification difficult or affecting the quality of the final product.

[0004] The dynamic stability control of enzymatic reaction process is also a difficult problem. Enzymatic reaction is usually accompanied by heat release and absorption, and enzyme activity is extremely sensitive to temperature. Traditional temperature control methods, such as heating or cooling through external jackets, are often difficult to achieve rapid and accurate regulation of the internal temperature of the reaction system due to their thermal inertia and the limitations of heat transfer efficiency. Temperature gradients are easily formed inside the reaction vessel, resulting in local overheating or low temperature zones. Local overheating may cause partial denaturation and inactivation of enzyme protein, while low temperature zones lead to a decrease in reaction rate. This unevenness of temperature distribution directly affects the overall efficiency and consistency of the reaction, and makes it difficult to ensure the repeatability of the reaction process.

[0005] Therefore, the existing enzymatic reaction system and method still have technical problems to be solved in terms of how to balance the efficiency of enzymatic reaction and the protection of enzyme activity, how to achieve dynamic stability control of the reaction system (especially temperature), and so on. These problems are interrelated and restrict the further improvement of the overall performance of enzymatic reaction process and the quality of the product. SUMMARY

[0006] An object of the present application is to solve at least the above problems and provide at least the advantages to be described later.

[0007] In order to achieve these objects and other advantages according to the present application, a kind of enzyme protein ultrasonic pretreatment and temperature-controlled enzyme acceleration reaction system is provided, comprising: Reaction container, which is internally provided with a bearing structure for accommodating enzyme and protein substrate, and is provided with an ultrasonic transducer array at the bottom of the reaction container; Ultrasonic generator, the output end of which is connected to the ultrasonic transducer array through wires, and the ultrasonic generator is configured to generate ultrasonic signals with a frequency of 20-40 kHz and a power density of 0.5-2 W / cm 2 ; Temperature sensor, which is embedded in the side wall of the reaction container, and the probe of the temperature sensor is arranged in the internal solution layer of the reaction container; Temperature control device, the input end of which is connected to the signal output end of the temperature sensor, and the temperature control device includes a heating element and a cooling circulation pipeline, the heating element is attached to the outer wall surface of the reaction container, and the cooling circulation pipeline surrounds the outer wall of the reaction container and communicates with an external refrigerator; Enzymatic reaction monitoring module, which includes a pH detection unit and a real-time turbidity sensor, and 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 container, and the enzymatic reaction monitoring module is configured to collect the pH value and turbidity data of the solution in real time; Controller, which is connected to the ultrasonic generator, the temperature control device and the enzymatic reaction monitoring module, and the controller is configured to control the ultrasonic generator to operate at a preset frequency for a preset time and then turn off; after turning off the ultrasonic generator, the temperature control device is controlled to adjust and maintain the temperature in the reaction container at 30-60 ℃; receive the turbidity data collected by the enzymatic reaction monitoring module, and output a termination signal to terminate the reaction when the turbidity data reaches a preset threshold.

[0008] Preferably, the controller is further configured to receive the pH data collected by the pH detection unit and calculate the change rate of the pH data; when the turbidity data reaches the preset threshold, it is verified whether the change rate of the pH data is within a preset change rate range, and only when the turbidity data reaches the preset threshold and the change rate of the pH data is within the preset change rate range, the termination signal is output to terminate the reaction.

[0009] Preferably, the controller is further configured to calculate the change rate of the turbidity data with time in real time, i.e. the turbidity change rate, and only when the following three conditions are met at the same time, the termination signal is output to terminate the reaction: Condition a, the turbidity data reaches a preset threshold; Condition b, the change rate of the pH data is within its preset change rate range; Condition c, the rate of change of turbidity is lower than a preset positive threshold.

[0010] Preferably, the controller is further configured to, when the controller outputs the termination signal, control the heating element of the temperature control device to raise the temperature in the reaction vessel to above 80℃ within 1 min and maintain the temperature for at least 2 min.

[0011] Preferably, the controller has pre-stored therein at least one database of combinations of ultrasonic parameters, the database of combinations of ultrasonic parameters associating different types of enzyme proteins with preferred combinations of ultrasonic frequency, power density and processing time; The controller is configured to receive user input or identify the current enzyme protein type information through a sensor, and call the corresponding ultrasonic frequency, power density and processing time from the database of combinations of ultrasonic parameters based on the current enzyme protein type information to control the ultrasonic generator to operate.

[0012] Preferably, it further comprises a stirring mechanism, which comprises: a drive motor fixed to the top or bottom of the reaction vessel; a stirring shaft connected to the output end of the drive motor at one end and extending to the internal solution layer of the reaction vessel, and a bearing structure fixed to the stirring shaft, the bearing structure being a mesh basket; The drive motor is electrically connected to the controller, and the controller is further configured to control the drive motor to operate at a first speed range during the operation of the ultrasonic generator; and control the drive motor to operate at a second speed range after the ultrasonic generator is turned off, the second speed range being lower than the first speed range.

[0013] Preferably, the mesh basket is composed of an inner mesh wall and an outer mesh wall, forming a hollow sandwich structure, the mesh diameter of the inner mesh wall is 50-200μm, and the mesh diameter of the outer mesh wall is 1-5mm, the mechanical strength of the outer mesh wall is higher than that of the inner mesh wall, and the mesh basket is provided with an openable and closable filling port on the side wall or the top cover.

[0014] Preferably, the side wall of the reaction vessel is provided with a sampling tube with a valve.

[0015] A method for operating an enzyme protein ultrasonic pretreatment and temperature-controlled enzyme acceleration reaction system is provided, comprising the following steps: S1, placing the enzyme and protein substrate in the bearing structure of the reaction vessel, and adding a buffer solution into the reaction vessel to completely immerse the bearing structure; S2, starting the ultrasonic generator by the controller, and making the ultrasonic transducer array operate according to the preset parameters for a preset time to perform ultrasonic pretreatment on the enzyme and protein substrate in the bearing structure; S3, after the ultrasonic generator is turned off, the temperature control device is controlled by the controller to adjust and maintain the temperature in the reaction container in a set temperature range required by the enzymatic reaction; S4, the pH value and turbidity data of the solution are collected in real time by the enzymatic reaction monitoring module, and the data are transmitted to the controller; S5, the controller outputs a termination signal to terminate the reaction based on the received turbidity data when the turbidity data reaches a preset threshold.

[0016] The present application at least includes the following beneficial effects: First, the present application systematically integrates ultrasonic pretreatment with specific parameters, precise temperature control and reaction endpoint judgment based on turbidity. The ultrasonic pretreatment step can effectively improve the structural accessibility of protein substrates at the physical level, creating more favorable initial conditions for subsequent enzymatic reactions. The integrated temperature control device overcomes the thermal inertia problem of traditional temperature control methods through rapid heating and cooling response, significantly improving the thermal stability of the reaction system. This multi-module synergy, from reaction initiation, process maintenance to endpoint judgment, effectively alleviates the inherent contradiction between enzymatic efficiency and enzyme activity protection, improving the controllability and efficiency of the entire reaction process.

[0017] Second, the present application introduces pH change rate as a second judgment parameter, forming a double-parameter verification mechanism with turbidity data, significantly improving the intelligence and reliability of reaction endpoint judgment. It can effectively distinguish between turbidity changes caused by real reaction progress and interference signals caused by bubbles, non-reactive particles or sensor noise. This cross-verification based on the essential characteristics of chemical reactions (pH change) and physical phenomena (turbidity change) reduces dependence on a single sensor and avoids premature or delayed termination caused by false positives, thereby more accurately capturing the reaction endpoint in complex reaction environments and further optimizing the quality and consistency of reaction products.

[0018] Third, based on the double-parameter verification, the present application further introduces turbidity change rate as a dynamic criterion, establishing a threefold verification logic combining static threshold and dynamic trend. It can sensitively capture the intrinsic feature of significantly reduced kinetic rate as the reaction approaches the endpoint, thereby improving the endpoint judgment from relying on instantaneous values to analyzing process trends at a higher level. This intelligent decision-making algorithm based on multi-parameter time sequence correlation characteristics can effectively filter transient interference without increasing hardware costs, greatly enhancing the robustness and accuracy of the system in judging the endpoint in complex environments.

[0019] 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.

[0020] 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.

[0021] 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

[0022] 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.

[0023] 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

[0024] 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.

[0025] 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.

[0026] like Figure 1As shown, the present embodiment provides an enzyme protein ultrasonic pretreatment and temperature-controlled enzyme acceleration reaction system, which mainly comprises: a reaction container 1, a bearing structure 2, an ultrasonic transducer array 3, an ultrasonic generator 4, a temperature sensor 5, a temperature control device 6, an enzyme reaction monitoring module and a controller 7.

[0027] The reaction container is a cylindrical container made of 316L stainless steel with a sealing cover, used for containing the buffer solution. The inside of the reaction container is provided with a bearing structure, which is a mesh basket made of 316L stainless steel in the present embodiment, used for containing the enzyme and protein substrate. The ultrasonic transducer array is fixedly installed on the outer wall of the bottom of the reaction container through waterproof sealing glue.

[0028] The ultrasonic generator is placed on the operating table beside the reaction container, and its output end is connected with the ultrasonic transducer array through shielding wires. The ultrasonic generator is configured to generate ultrasonic signals with adjustable frequency in the range of 20-40 kHz and adjustable power density in the range of 0.5-2 W / cm 2 .

[0029] The temperature sensor is a Pt100 platinum resistance temperature sensor, and its sensing probe is embedded in the side wall of the reaction container and extends into the internal solution layer of the reaction container 1, used for directly measuring the solution temperature.

[0030] The temperature control device includes a silicone heating film (i.e. heating element) attached to the outer wall surface of the reaction container, and a purple copper pipeline (i.e. cooling circulation pipeline) coiled on the outer wall of the reaction container. The cooling circulation pipeline is in communication with an external circulating water chiller (i.e. external chiller 14) through an insulating pipeline. The signal output end of the temperature sensor is connected to the input end of the temperature control device.

[0031] The enzyme 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). The electrode 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 container through the opening of the reaction container top cover 13. The enzyme reaction monitoring module is configured to collect the pH value and turbidity NTU data of the solution once per second.

[0032] The controller is an embedded industrial computer, which establishes communication connection with the ultrasonic generator, the temperature control device and the enzyme reaction monitoring module through data lines.

[0033] Effect of ultrasonic pretreatment on enzyme hydrolysis: The present inventors believe that the ultrasonic pretreatment step with specific parameters (frequency 20-40 kHz, power density 0.5-2.0 W / cm 2, time 5-15 min) can significantly improve the efficiency of subsequent enzymatic reaction, and the main principle is based on the following aspects: The cavitation effect produced by ultrasonic wave in liquid medium is its main mechanism. This effect can produce extremely high instantaneous pressure and temperature in the local area, and produce strong mechanical shear force on the protein substrate. This effect can effectively destroy the tight higher structure of the protein substrate, partially denature and stretch it, and expose more enzyme cutting sites originally embedded in the molecule, thereby significantly increasing the accessible area and reaction probability of the enzyme and the substrate; The micro-jet and strong vibration produced by ultrasonic wave in the solution can significantly intensify the mass transfer process in the reaction system. This not only promotes the diffusion and collision frequency of enzyme molecules and substrate molecules, but also helps to break the liquid film resistance at the reaction interface, making the enzymatic reaction more rapid and uniform; Within a moderate parameter range, ultrasonic wave may produce a certain perturbation effect on the enzyme protein molecules, induce a moderate change in the conformation of the enzyme molecules that is conducive to catalytic reaction, thereby activating the enzyme molecules to some extent, improving their instantaneous catalytic efficiency, and avoiding the risk of irreversible inactivation of the enzyme caused by high-intensity ultrasonic wave.

[0034] Therefore, through the synergistic effect of the above-mentioned multiple physical effects, the ultrasonic pretreatment step creates a more favorable initial condition for the subsequent enzymatic reaction, making the reaction start faster and the process deeper, thereby macroscopically showing a significant shortening of the enzymatic reaction time and an effective improvement in the final product yield. The parameter range selected by the present application is an optimized balance between fully stimulating the above-mentioned beneficial effects and avoiding damage to enzyme activity.

[0035] Experimental group: the system of the present embodiment is used, and the specific operation is as follows: The trypsin and casein substrate are placed in the mesh basket, and the phosphate buffer solution (pH=7.8) is added to the reaction container to completely immerse the carrying structure. The ultrasonic pretreatment parameters are set by the controller: frequency 25 kHz, power density 1.0 W / cm 2 , treatment time 10 min. After the pretreatment is completed, the system automatically turns off the ultrasonic generator, and the reaction system temperature is controlled at 50°C for enzymatic reaction. The turbidity is monitored in real time by the enzymatic reaction monitoring module, and when the turbidity value reaches 1500 NTU, the controller outputs a termination signal, and the system performs heat inactivation to terminate the reaction.

[0036] Control group: except that ultrasonic pretreatment is not performed, the other conditions (including the types and amounts of enzyme and substrate, buffer solution, reaction temperature 50°C, termination conditions, etc.) are exactly the same as those of the experimental group. The enzyme and substrate are directly placed in a 50°C environment to start the enzymatic reaction.

[0037] The product yield and enzymolysis rate of two groups of experiments are determined and compared: the product yield of the experimental group (pretreated by ultrasonic wave) is increased by 15.2% compared with the control group. The time required for the experimental group to reach the termination condition (turbidity 1500 NTU) from the start of the enzymolysis reaction is 45 min, while the time required for the control group to reach the same termination condition is 72 min. The enzymolysis rate of the experimental group is significantly higher than that of the control group. The results show that the system of the present application can effectively improve the efficiency of the enzymolysis reaction by introducing the ultrasonic pretreatment step with specific parameters, which is specifically reflected in significantly shortening the reaction time and improving the final product yield.

[0038] In another embodiment of the present application, a method for judging the end point of an enzymolysis reaction is provided, which further introduces the pH change rate as a cooperative criterion on the basis of real-time monitoring of turbidity data, and constructs a double-parameter verification mechanism to solve the technical problem of single turbidity criterion being easily disturbed and leading to end point misjudgment. The following examples are used to illustrate the method: Example 1: The method for judging the end point of an enzymolysis reaction based on double-parameter verification adopts a turbidity data and pH change rate double-parameter cooperative verification mechanism, which includes: A reaction container is provided with a mesh basket carrying enzymes and protein substrates. An ultrasonic transducer array is arranged at the bottom of the reaction container.

[0039] An enzymolysis reaction monitoring module includes: A pH detection unit adopts a composite pH electrode; a real-time turbidity sensor adopts an insertion type turbidity probe; and a controller adopts an embedded industrial computer.

[0040] Implementation steps: (1) Place trypsin and casein substrate in the mesh basket, and add phosphate buffer solution to completely immerse; (2) Start ultrasonic pretreatment: frequency 28 kHz, power density 1.2 W / cm 2 , treatment time 8 min; (3) Turn off the ultrasonic wave, and maintain the temperature of the reaction system at 37℃; (4) The controller executes the following judgment logic: Real-time acquisition: turbidity value T (NTU), pH value; Real-time calculation: pH change rate R = ΔpH / Δt (pH / min); Judgment condition: if T ≥ T set and R min ≤ R ≤ R max , output termination signal, otherwise continue the reaction; Wherein: the turbidity preset threshold T set = 1800 NTU; the pH change rate preset range R min = -0.015 pH / min, Rmax = 0.015 pH / min; sampling interval Δt = 1 s; the pH change rate is calculated by a sliding window method, and the window width is 10 sampling points.

[0041] The experimental group (Example 1) uses the double-parameter verification method of the embodiment, and the control group only uses the single parameter of turbidity to judge. The results show that the experimental group has accurate reaction termination timing, the product yield is increased by 12.3%, and the enzyme activity loss is reduced by 15.6%. The control group has 2 misjudgments due to bubble interference, and the product yield fluctuates greatly.

[0042] Example 2: Parameter optimization suitable for acid protease. On the basis of Example 1, parameter optimization is carried out according to the characteristics of acid protease: Reaction temperature: 45℃; turbidity preset threshold: 2200 NTU; pH change rate preset range: -0.008 pH / min to 0.008 pH / min; buffer system: citric acid-sodium phosphate dibasic buffer.

[0043] The present application effectively distinguishes the true reaction endpoint from the sensor interference signal by taking the pH change rate as the second judgment parameter and forming complementary verification with the turbidity data. This double-parameter verification mechanism significantly improves the accuracy and reliability of reaction endpoint judgment, solving the technical problem of single turbidity sensor being easily disturbed.

[0044] In another embodiment of the present application, an enzyme hydrolysis reaction endpoint judgment method is provided, which further introduces the turbidity change rate as a third criterion on the basis of double-parameter verification, and constructs a triple verification mechanism based on the combination of static threshold and dynamic trend, aiming to more accurately identify the reaction kinetics endpoint and improve the robustness of judgment in a complex interference environment. The following examples are used to illustrate the specific embodiments: Example 3: Triple-parameter endpoint judgment based on turbidity threshold, pH change rate and turbidity change rate, on the basis of Example 1 or 2, a triple verification mechanism is constructed by introducing the turbidity change rate as a third judgment parameter, and in the configuration, the controller is further configured to perform the following processing logic: Real-time acquisition of turbidity data and pH data; Real-time calculation of pH change rate R pH = ΔpH / Δt; Real-time calculation of turbidity change rate R Turb = ΔT urb / Δt; Based on the following three conditions for collaborative judgment.

[0045] The judgment conditions include: Condition a: turbidity data ≥ preset turbidity threshold T set ; Condition b: pH change rate RpH within a preset range [R pHmin , R pHmax ]; condition c: rate of turbidity change R Turb ≤ preset positive threshold R Turbmax .

[0046] Only when conditions a, b, and c are met simultaneously, the controller outputs a termination signal to terminate the reaction.

[0047] In one embodiment: 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 = 1 s; change rate calculation all use moving average method, window width is 10 sampling points.

[0048] By introducing the turbidity change rate, a dynamic parameter, the kinetic characteristics of the reaction approaching the end point can be effectively identified. When the reaction substrate is substantially depleted, the turbidity change rate will be significantly reduced and stabilized. This parameter can effectively distinguish the true reaction end point from the transient turbidity fluctuations caused by bubbles, particle aggregation or sensor noise, thus providing more reliable end point judgment in complex reaction environment.

[0049] The present application is not limited to the above-mentioned specific parameter values, and those skilled in the art can adjust the above-mentioned threshold values and ranges according to the characteristics of different enzyme-substrate systems, and these adjustments still belong to the protection scope of the present application.

[0050] In another embodiment of the present application, the system further comprises a termination execution module electrically connected to the controller. The termination execution module is not an independent hardware device, but is realized by the controller calling the existing temperature control device of the system through programming logic to realize its function. Specifically, when the controller outputs a termination signal according to the preset judgment logic (such as based on turbidity data, or a multi-parameter verification mechanism), the signal will trigger the controller to execute the predetermined termination program.

[0051] In the process of enzymatic reaction, when the controller determines that the reaction reaches the end point and outputs a termination signal, the controller immediately controls the heating element (such as the silicone heating film mentioned above) of the temperature control device to enter a high-intensity working mode. Specifically, the controller controls the heating element to rapidly raise the temperature of the solution in the reaction vessel from the enzymatic reaction temperature (for example, 30-60℃) to 85℃ within 1 min, and maintains the temperature for 3 min. After this high-temperature maintenance stage, the system can automatically stop heating, or the controller can start the cooling circulation pipeline to cool down, thereby completing the termination operation of the entire enzymatic reaction.

[0052] The present embodiment uses the temperature control device inherent to the system as a termination execution module, and sets its specific working mode, thereby realizing rapid, complete and physical termination of the enzymatic reaction without introducing external chemicals. This scheme ensures that the reaction can be terminated in time and effectively at the most appropriate time determined by the controller, avoiding excessive enzymolysis or degradation of the target product due to termination delay, and eliminating the problem of product contamination caused by the addition of chemical termination agents.

[0053] In another embodiment of the present application, the controller has pre-stored therein at least one ultrasonic parameter combination database. The database is stored in the form of a data table, which records the association of different types of enzyme proteins with the preferred parameter combinations of ultrasonic frequency, power density and treatment time.

[0054] Before the system starts the ultrasonic pretreatment program, the controller is configured to receive the current enzyme protein type information input by the user through the human-machine interface (for example, selecting "trypsin" or "acid protease"). Alternatively, in another optional implementation, the system can be equipped with a simple biosensor for automatically identifying and feeding back the current enzyme protein type information to the controller.

[0055] After the controller obtains the current enzyme protein type information, it immediately queries the ultrasonic parameter combination database based on this information and calls the preferred ultrasonic frequency, power density and treatment time corresponding thereto. For example, when the current enzyme protein type is "trypsin", the controller calls and sets the ultrasonic frequency to 28 kHz, the power density to 1.2 W / cm 2 and the treatment time to 8 min from the database. The controller controls the ultrasonic generator to operate according to the parameters called by this set, and completes the targeted ultrasonic pretreatment step.

[0056] The present embodiment introduces an ultrasonic parameter combination database associated with the type of enzyme protein, so that the system can tailor the pretreatment scheme for different enzyme proteins. This solves the technical problem that a fixed ultrasonic parameter range may not be suitable for the characteristics of diversified enzyme proteins, and realizes the individualization and intelligentization of the pretreatment stage. By maximizing the pretreatment efficiency of ultrasonic waves on a specific enzyme-substrate system while avoiding damage to enzyme activity, the final enzymatic reaction effect is optimized and stabilized at a deeper level, thereby improving the applicability and reproducibility of the processing effect of the system.

[0057] In another embodiment of the present application, the system further comprises a stirring mechanism. The stirring mechanism specifically comprises: a driving motor 10 fixed to the top of the reaction container through a support; 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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: 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. 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; 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℃. 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 when the turbidity data reaches a preset threshold, based on the received turbidity data.

[0066] 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.

[0067] 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 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.

2. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 1, characterized in that, 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.

3. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 2, characterized in that, 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 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.

4. 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.

5. 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 preferred 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.

6. 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 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.

7. The enzyme protein ultrasonic pretreatment and temperature-controlled enzyme accelerated reaction system as described in claim 6, 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.

8. 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.

9. 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 8, 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 the data is transmitted to the controller. 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.

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