Preparation device and method for bio-imprinting enzyme

Abstract

The invention relates to the technical field of bio-imprinting enzyme preparation, and discloses a bio-imprinting enzyme preparation device and method.The device comprises a shell, a control panel is arranged on the outer side of the shell, a partition plate is fixedly connected to the inner wall of the shell, and a reaction combination mechanism is arranged at the top of the shell; a solvent gradient conversion mechanism is arranged at the top of the partition plate, the reaction combination mechanism is connected with the solvent gradient conversion mechanism, an enzyme liquid treatment and recovery mechanism is arranged on the inner bottom wall of the shell, the solvent gradient conversion mechanism is connected with the enzyme liquid treatment and recovery mechanism, and a pH adjusting mechanism is arranged on the inner side wall of the shell. By adopting the technical scheme that the feeding pump I and the feeding pump II are matched with the coiled pipe in the double-layer constant-temperature reaction box, full contact and uniform flow of an enzyme solution and an inhibitor solution are realized, and the acid-base dripping amount is adjusted through the pH sensor and the injection pump, so that the stability of reaction conditions and the optimal combination efficiency are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of bio-imprinted enzymes, and in particular to a device and method for preparing bio-imprinted enzymes. Background Art

[0002] Bioimprinted enzymes are natural enzymes modified through bioengineering or chemical modification to impart specific functions or enhance their inherent properties. Currently, while some methods for bioimprinted enzyme preparation are capable of isolating and purifying enzymes, they often suffer from complex processes, low efficiency, and insufficient protection of enzyme activity.

[0003] In the prior art, due to the lack of precise control over the reaction process, the binding efficiency of the enzyme and the inhibitor is low, which often leads to poor consistency of the product. At the same time, during the solvent gradient conversion process, the solvent ratio adjustment is not accurate enough, which may cause the enzyme solution to be insufficiently mixed, thereby affecting the reaction effect. In addition, during the impurity removal process, the cleaning step usually adopts a single or simple operation mode, which cannot effectively remove the inhibitors and other impurities remaining on the enzyme surface, resulting in the difficulty in improving the purity of the finished enzyme. For the activity protection of the enzyme, the prior art often ignores the necessity of a low-temperature environment, and the three-dimensional conformation of the enzyme is easily damaged during operation, which directly affects the activity and stability of the product.

[0004] In addition, in terms of separating waste liquid from finished enzyme solution, existing devices usually rely on manual operation or simple physical methods, which makes it difficult to achieve precise diversion and easily causes mixing of waste liquid and finished enzyme solution, increasing the difficulty of subsequent separation. At the same time, the lack of automated control means makes the entire preparation process highly dependent on manual labor, with low production efficiency and poor operational stability. Overall, the existing technology has significant deficiencies in reaction uniformity, solvent conversion, impurity removal, enzyme activity protection, and automated operation.

[0005] Therefore, the present invention proposes a device and method for preparing bio-imprinted enzymes to address the deficiencies of the prior art. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides a device and method for preparing bio-imprinted enzymes, which solve the problems of the existing technology in terms of reaction uniformity, solvent conversion, impurity removal, enzyme activity protection and automated operation.

[0007] To achieve the above object, the present invention is realized through the following technical solutions: A preparation device for bioimprinting enzymes, including a housing, a control panel is arranged on the outer side of the housing, a partition is fixedly connected to the inner wall of the housing, a reaction combination mechanism is arranged on the top of the housing, a solvent gradient conversion mechanism is arranged on the top of the partition, the reaction combination mechanism is connected to the solvent gradient conversion mechanism, an enzyme solution treatment and recovery mechanism is arranged on the inner bottom wall of the housing, the solvent gradient conversion mechanism is connected to the enzyme solution treatment and recovery mechanism, a pH adjustment mechanism is arranged on the inner side wall of the housing, and the pH adjustment mechanism is connected to the reaction combination mechanism;

[0008] The reaction combination mechanism includes an enzyme solution storage bottle and an inhibitor solution storage bottle. Both the enzyme solution storage bottle and the inhibitor solution storage bottle are fixedly connected to the top of the housing. A first feed pump and a second feed pump are respectively installed on the top of the housing. The input end of the first feed pump is communicated with the enzyme solution storage bottle through a pipeline, and the input end of the second feed pump is connected to the inhibitor solution storage bottle through a pipeline. A double-layer constant temperature reaction box is fixedly connected to the inner side wall of the housing. A serpentine tube is arranged inside the double-layer constant temperature reaction box. The output end of the first feed pump is communicated with the serpentine tube through a pipeline, and the output end of the second feed pump is communicated with the serpentine tube through a pipeline. A control valve is arranged on the outer side of the serpentine tube.

[0009] Preferably, the solvent gradient conversion mechanism includes a second mixing tank and a gradient solvent pump. The second mixing tank is fixedly connected to the top of the partition. A second cooling jacket is arranged on the outer side of the second mixing tank. The gradient solvent pump is installed at the bottom of the double-layer constant temperature reaction box. The input end of the gradient solvent pump is connected to the end of the serpentine tube passing through the outer side of the double-layer constant temperature reaction box. The output end of the gradient solvent pump is connected to three first mixing tanks through pipelines. A first cooling jacket is arranged on the outer side of the first mixing tank. The first mixing tank close to the partition side is connected to the second mixing tank through a pipeline, and a control valve is arranged on the outer side of the pipeline. A first mixing component is arranged inside the second mixing tank, and the first mixing component is used to mix the enzyme solution inside the second mixing tank.

[0010] Preferably, the first mixing component includes a first motor. The output end of the first motor is fixedly connected to a first connecting rod. A plurality of first mixing rods are fixedly connected to the outer side of the first connecting rod. A first mixing head is fixedly connected to the bottom of the first connecting rod.

[0011] Preferably, the enzyme solution treatment and recovery mechanism includes a low-temperature freezing tank fixedly connected to the inner bottom wall of the outer shell. A liquid nitrogen device is arranged outside the low-temperature freezing tank. The low-temperature freezing tank is communicated with the second mixing tank through a pipeline, and a control valve is arranged outside the pipeline. On one side of the inner bottom wall of the outer shell, a finished enzyme solution collection tank is fixedly connected, and on the other side of the inner bottom wall of the outer shell, a waste liquid collection tank is fixedly connected. Both the finished enzyme solution collection tank and the waste liquid collection tank are communicated with the low-temperature freezing tank through pipelines, and control valves are arranged outside the pipelines. On the inner bottom wall of the outer shell, a first spraying tank, a second spraying tank, and a third spraying tank are fixedly connected. The first spraying tank, the second spraying tank, and the third spraying tank are all communicated with the low-temperature freezing tank through pipelines, and control valves are arranged outside the pipelines. A second mixing component is arranged inside the low-temperature freezing tank for mixing the enzyme solution inside the low-temperature freezing tank. A diversion component is arranged on the inner wall of the low-temperature freezing tank for diverting the enzyme solution. A connecting plate is arranged between the second mixing component and the diversion component. A pipeline is communicated at the bottom of the connecting plate, and a control valve is arranged outside the pipeline. The connecting plate is fixedly connected to the inner wall of the low-temperature freezing tank. A turbidity sensor is arranged on the inner wall of the low-temperature freezing tank.

[0012] Preferably, the second mixing component includes a second motor. The output end of the second motor is fixedly connected with a second connecting rod, and the bottom of the second connecting rod is fixedly connected with a second mixing head.

[0013] Preferably, the diversion component includes a first diversion plate and a second diversion plate. The first diversion plate is fixedly connected to the inner wall of the outer shell, and the second diversion plate is fixedly connected to the inner wall of the outer shell. The turbidity sensor is arranged between the first diversion plate and the second diversion plate.

[0014] Preferably, the pH adjustment mechanism includes a pH controller installed on the inner side wall of the outer shell. The pH controller is connected with a pH sensor, and the detection part of the pH sensor is located inside the serpentine tube. An injection pump one and an injection pump two are respectively installed on the inner side wall of the outer shell. The input end of the injection pump one is communicated with an acid solution storage bottle through a pipeline, and the input end of the injection pump two is communicated with an alkali solution storage bottle through a pipeline. The output end of the injection pump one is communicated with the serpentine tube through a pipeline, and the output end of the injection pump two is communicated with the serpentine tube through a pipeline.

[0015] Preferably, the reaction combination mechanism further includes a detector installed on the inner top wall of the outer shell, and the detection head of the detector is located outside the serpentine tube.

[0016] Preferably, the solvent gradient conversion mechanism further includes a laser Raman spectrometer installed on the top of the partition board, and the detection head of the laser Raman spectrometer is located inside the pipeline connecting the second mixing tank and the low-temperature freezing tank.

[0017] The present invention also provides a preparation method for a bioimprinted enzyme, comprising the following steps:

[0018] S1. Add the enzyme solution to the enzyme solution storage bottle, and add the inhibitor solution to the inhibitor solution storage bottle. Start feeding pump 1 and feeding pump 2, and respectively transport the enzyme solution and the inhibitor solution to the serpentine tube of the double-layer constant-temperature reaction box through pipelines to provide a uniformly flowing basic solution for subsequent reactions;

[0019] S2. The enzyme and the inhibitor fully contact through the serpentine tube in the double-layer constant-temperature reaction box, and a binding reaction is carried out under constant-temperature conditions. The pH sensor monitors the pH value of the solution in real time, and the pH controller adjusts the dropping amounts of the acid solution and the base solution through injection pump 1 and injection pump 2 to ensure that the reaction is within the optimal pH range. The binding efficiency is monitored by a detector, and after reaching the set requirements, proceed to the next step;

[0020] S3. The combined enzyme-inhibitor complex solution is successively fed into three mixing tanks 1 and mixing tank 2 through a gradient solvent pump. Mixing assembly 1 completes the full mixing of the solution in mixing tank 2. Cooling jacket 1 and cooling jacket 2 maintain a low-temperature environment. The gradient solvent pump gradually adjusts the ratio of the aqueous solution to the lipophilic solvent, and a laser Raman spectrometer is used for detection;

[0021] S4. The solution after solvent gradient conversion enters a low-temperature freezing tank through a pipeline. The liquid nitrogen device maintains the temperature of the low-temperature freezing tank at -10°C. Then, through spray tank 1, a weakly polar solvent is sprayed to remove non-polar impurities in the enzyme solution. Through spray tank 2, a strongly polar solvent is sprayed to remove polar inhibitors on the surface of the enzyme. Through spray tank 3, an inert solvent is sprayed to finally wash and neutralize the enzyme solution to ensure its purity;

[0022] S5. Mixing assembly 2 ensures the uniform distribution and dynamic flow of the enzyme solution, so that the three-dimensional conformation of the enzyme is "frozen" in a low-temperature environment, ensuring the high activity and stability of the enzyme;

[0023] S6. For the enzyme solution that has been mixed and sprayed, the enzyme solution is discharged by opening the control valve of the pipeline on the connecting plate. The deflector is designed to be inclined to guide the enzyme solution to flow downward. Since the waste liquid contains a large amount of inhibitor residues and impurities, these components usually have a higher density than the finished enzyme solution. The waste liquid with a high density is more likely to settle to the bottom of the low-temperature freezing tank under the action of gravity and deflectors 1 and 2. When the waste liquid and the finished enzyme solution flow through the diversion area, the turbidity sensor detects the purity of the liquid in real time. If the turbidity is high, it is determined as waste liquid, and the control valve on the pipeline connecting the low-temperature freezing tank to the waste liquid collection tank is opened to discharge it to the waste liquid collection tank. If the turbidity is low, it is determined as the finished enzyme solution, and the control valve on the pipeline connecting the low-temperature freezing tank to the finished enzyme solution collection tank is opened. In the finished enzyme solution collection tank, the waste liquid and the finished enzyme are controlled to be separated by independent control valves to avoid mixing or contamination;

[0024] S7. The whole process is automatically managed by a PLC control system. Key parameters such as flow rate, temperature, and pH value are dynamically adjusted through the control panel. The system records and feeds back various data, optimizes the reaction conditions in real time, ensures the stable and efficient operation of the device, and provides a basis for subsequent process improvement.

[0025] The present invention provides a device and method for preparing a bioimprinted enzyme, which has the following beneficial effects:

[0026] 1. The present invention adopts the technical scheme of combining feed pump 1 and feed pump 2 with the serpentine tube in the double-layer constant-temperature reaction box, realizing the full contact and uniform flow of the enzyme solution and the inhibitor solution, and adjusting the acid-base dropping amount through the pH sensor and the injection pump, ensuring the stability of the reaction conditions and the best binding efficiency. Compared with the prior art lacking precise pH control and uniform mixing, the problems of low reaction efficiency and insufficient product consistency are solved.

[0027] 2. The present invention adopts the technical scheme of combining a gradient solvent pump and a mixing tank, and cooperates with real-time detection by a laser Raman spectrometer to achieve precise adjustment of the solvent gradient conversion, and maintains a low-temperature environment through a cooling jacket, ensuring the stability and activity of the enzyme solution structure. Compared with the prior art with uneven solvent mixing or insufficient temperature control accuracy, the problems of unstable solvent conversion process and loss of enzyme activity are solved.

[0028] 3. The present invention adopts the technical scheme of spraying weak polar, strong polar, and inert solvents step by step with a spray tank, gradually removing non-polar impurities and polar inhibitors in the enzyme solution, and combining with a low-temperature freezing environment to freeze and stabilize the three-dimensional conformation of the enzyme. Compared with the prior art with incomplete impurity removal or damaged enzyme conformation during the cleaning process, the problems of insufficient purity of the finished enzyme and loss of activity are solved.

[0029] 4. The present invention adopts the technical scheme of combining a deflector and a turbidity sensor. By real-time detecting the liquid purity, the outlet of the finished enzyme solution and the waste liquid is switched by an independent control valve, and the whole process is automatically and dynamically controlled by the PLC system. Compared with the prior art lacking real-time detection and inaccurate shunting, the problems of low separation efficiency and contamination of the finished enzyme solution are solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a perspective view of the present invention;

[0031] Figure 2 is a front view of the present invention;

[0032] Figure 3 is a side sectional view of the present invention;

[0033] Figure 4Schematic structural diagram of the pH adjustment mechanism of the present invention;

[0034] Figure 5 Schematic structural diagram of the solvent gradient conversion mechanism of the present invention;

[0035] Figure 6 Schematic structural diagram of the second mixing tube of the present invention;

[0036] Figure 7 Schematic structural diagram of the first mixing tank of the present invention;

[0037] Figure 8 Schematic structural diagram of the enzyme solution treatment and recovery mechanism of the present invention;

[0038] Figure 9 Schematic internal structure diagram of the low-temperature freezing tank of the present invention.

[0039] Among them, 1. Outer shell; 2. Control panel; 3. Partition board; 4. Reaction combination mechanism; 401. Enzyme solution storage bottle; 402. Inhibitor solution storage bottle; 403. Feed pump one; 404. Feed pump two; 405. Double-layer constant temperature reaction box; 406. Coiled tube; 407. Detector; 5. Solvent gradient conversion mechanism; 501. First mixing tank; 502. First cooling jacket; 503. Second mixing tank; 504. Second cooling jacket; 505. Gradient solvent pump; 506. Laser Raman spectrometer; 507. Motor one; 508. Connecting rod one; 509. First mixing head; 5010. First mixing rod; 6. Enzyme solution treatment and recovery mechanism; 601. Low-temperature freezing tank; 602. Liquid nitrogen device; 603. First spray tank; 604. Second spray tank; 605. Third spray tank; 606. Finished enzyme solution collection tank; 607. Turbidity sensor; 608. Waste liquid collection tank; 609. Motor two; 6010. Connecting rod two; 6011. Second mixing head; 6012. Connecting plate; 6013. First deflector; 6014. Second deflector; 7. pH adjustment mechanism; 701. pH controller; 702. pH sensor; 703. Acid solution storage bottle; 704. First injection pump; 705. Alkali solution storage bottle; 706. Second injection pump. Specific embodiments

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Please refer to Figures 1-9, an embodiment of the present invention provides a preparation device for bioimprinted enzymes, which includes a housing 1. A control panel 2 is arranged on the outer side of the housing 1. A partition 3 is fixedly connected to the inner wall of the housing 1. A reaction combination mechanism 4 is arranged on the top of the housing 1. A solvent gradient conversion mechanism 5 is arranged on the top of the partition 3. The reaction combination mechanism 4 is connected to the solvent gradient conversion mechanism 5. An enzyme solution treatment and recovery mechanism 6 is arranged on the inner bottom wall of the housing 1. The solvent gradient conversion mechanism 5 is connected to the enzyme solution treatment and recovery mechanism 6. A pH adjustment mechanism 7 is arranged on the inner side wall of the housing 1. The pH adjustment mechanism 7 is connected to the reaction combination mechanism 4.

[0042] Specifically, the housing 1 serves as an overall support structure, providing a stable installation space for each internal mechanism; the control panel 2 on the outer side facilitates the operator to set parameters and perform real-time control on the device, improving the operation convenience; the partition 3 reasonably divides the internal space of the device, optimizing the layout of each mechanism; the reaction combination mechanism 4 enables the enzyme solution and the inhibitor solution to fully contact and react under specific conditions, laying the foundation for the formation of bioimprinted enzymes; the solvent gradient conversion mechanism 5 can gradually adjust the solvent ratio, effectively process the reaction solution, and ensure the smooth progress of subsequent steps; the enzyme solution treatment and recovery mechanism 6 realizes the fine treatment of the enzyme solution and the effective separation of waste liquid and finished products, improving the product purity and raw material utilization rate; the pH adjustment mechanism 7 monitors and adjusts the pH value during the reaction in real time, ensuring that the reaction is always carried out in the best environment, which is beneficial to improving the reaction efficiency and the quality of bioimprinted enzymes.

[0043] Please refer to Figures 1-4 , the reaction combination mechanism 4 includes an enzyme solution storage bottle 401 and an inhibitor solution storage bottle 402. Both the enzyme solution storage bottle 401 and the inhibitor solution storage bottle 402 are fixedly connected to the top of the housing 1. A feed pump one 403 and a feed pump two 404 are respectively installed on the top of the housing 1. The input end of the feed pump one 403 is communicated with the enzyme solution storage bottle 401 through a pipeline. The input end of the feed pump two 404 is connected to the inhibitor solution storage bottle 402 through a pipeline. A double-layer constant temperature reaction box 405 is fixedly connected to the inner side wall of the housing 1. A serpentine tube 406 is arranged inside the double-layer constant temperature reaction box 405. The output end of the feed pump one 403 is communicated with the serpentine tube 406 through a pipeline. The output end of the feed pump two 404 is communicated with the serpentine tube 406 through a pipeline. A control valve is arranged on the outer side of the serpentine tube 406.

[0044] Specifically, through the enzyme solution reservoir 401, the enzyme solution can be stably stored. In cooperation with the first feeding pump 403, after the first feeding pump 403 is started, it extracts the enzyme solution from the enzyme solution reservoir 401 and transports it through a pipeline to the serpentine tube 406 inside the double-layer constant-temperature reaction box 405, achieving the precise and stable supply of the enzyme solution to the reaction area; through the inhibitor solution reservoir 402, the inhibitor solution is stably stored. In cooperation with the second feeding pump 404, after the second feeding pump 404 is started, it extracts the inhibitor solution from the inhibitor solution reservoir 402 and transports it through a pipeline to the serpentine tube 406 inside the double-layer constant-temperature reaction box 405, achieving the precise and stable supply of the inhibitor solution to the reaction area; through the double-layer constant-temperature reaction box 405, a stable internal temperature environment is maintained. In cooperation with the serpentine tube 406, the enzyme solution and the inhibitor solution in the serpentine tube 406 react under constant-temperature conditions, providing suitable temperature conditions for the binding reaction of the enzyme and the inhibitor and ensuring the stability and consistency of the reaction; through the control valve, installed on the outer side of the serpentine tube 406 and cooperating with the serpentine tube 406, it can control the flow state of the solution in the tube, be flexibly opened or closed, facilitating the regulation of the reaction process and ensuring that the reaction proceeds according to the set process.

[0045] Please refer to Figure 1 、 Figure 2 、 Figure 3 、 Figure 5 and Figure 7

[0046] , the solvent gradient conversion mechanism 5 includes a second mixing tank 503 and a gradient solvent pump 505. The second mixing tank 503 is fixedly connected to the top of the partition plate 3. A second cooling jacket 504 is arranged on the outer side of the second mixing tank 503. The gradient solvent pump 505 is installed at the bottom of the double-layer constant-temperature reaction box 405. The input end of the gradient solvent pump 505 is communicated with one end of the serpentine tube 406 passing through the outer side of the double-layer constant-temperature reaction box 405. The output end of the gradient solvent pump 505 is connected with three first mixing tanks 501 through a pipeline. A first cooling jacket 502 is arranged on the outer side of the first mixing tank 501. The first mixing tank 501 close to the partition plate 3 side is communicated with the second mixing tank 503 through a pipeline, and a control valve is arranged on the outer side of the pipeline.

[0046] Specifically, the mixing tank II 503 provides an accommodation space for the mixing and subsequent treatment of solvents. In cooperation with the cooling jacket II 504, the cooling function of the cooling jacket II 504 is used to cool the solution in the tank, realizing the mixing and reaction of solvents in a low-temperature environment and avoiding the influence of excessive temperature on the properties of solvents and the reaction effect. Through the gradient solvent pump 505, as a power transmission component, in cooperation with the serpentine tube 406, the solution in the serpentine tube 406 is sucked out and delivered to the three mixing tanks I 501 in cooperation with the three mixing tanks I 501, realizing the transfer and distribution of the solution between different treatment units and ensuring the continuity and orderliness of the solution gradient conversion process. Through the three mixing tanks I 501, multiple treatment units are provided for the gradient conversion of the solution. In cooperation with the cooling jacket I 502, the cooling function of the cooling jacket I 502 is used to cool the solution in the tank, providing a suitable low-temperature environment for the gradient conversion of solvents, which helps to realize the gradient conversion operation of the solution. At the same time, in cooperation with the pipeline and control valve near the baffle 3 side, the flow direction of the solution can be controlled, realizing the transfer and precise control of the solution at different stages and ensuring the accuracy and stability of the solvent gradient conversion process.

[0047] Please refer to Figure 6 , a mixing component I is arranged inside the mixing tank II 503. The mixing component I is used to mix the enzyme solution inside the mixing tank II 503. The mixing component I includes a motor I 507. The output end of the motor I 507 is fixedly connected with a connecting rod I 508. A plurality of mixing rods I 5010 are fixedly connected to the outer side of the connecting rod I 508. The bottom of the connecting rod I 508 is fixedly connected with a mixing head I 509.

[0048] Specifically, through the motor I 507, as a power source, in cooperation with the connecting rod I 508, after the motor I 507 is powered on and operates, it drives the connecting rod I 508 to rotate at a high speed, realizing a stable and continuous power output for the mixing operation and ensuring the efficient progress of the mixing process. Through the connecting rod I 508, one end is connected to the motor I 507, and the other end is connected to the mixing rod I 5010 and the mixing head I 509. In cooperation with the motor I 507, the mixing rod I 5010 and the mixing head I 509, under the drive of the motor I 507, the rotational power is transmitted to the mixing rod I 5010 and the mixing head I 509, realizing all-round and multi-angle mixing actions, making the mixing of the enzyme solution in the mixing tank II 503 more sufficient.

[0049] Please refer to Figure 1 , Figure 2 , Figure 3 , Figure 8 and Figure 9, the enzyme solution treatment and recovery mechanism 6 includes a low-temperature freezing tank 601, which is fixedly connected to the inner bottom wall of the outer shell 1. A liquid nitrogen device 602 is arranged outside the low-temperature freezing tank 601. The low-temperature freezing tank 601 is connected to the second mixing tank 503 through a pipeline, and a control valve is arranged outside the pipeline. On one side of the inner bottom wall of the outer shell 1, a finished enzyme solution collection tank 606 is fixedly connected. On the other side of the inner bottom wall of the outer shell 1, a waste liquid collection tank 608 is fixedly connected. Both the finished enzyme solution collection tank 606 and the waste liquid collection tank 608 are connected to the low-temperature freezing tank 601 through pipelines, and control valves are arranged outside the pipelines. On the inner bottom wall of the outer shell 1, a first spray tank 603, a second spray tank 604, and a third spray tank 605 are fixedly connected. The first spray tank 603, the second spray tank 604, and the third spray tank 605 are all connected to the low-temperature freezing tank 601 through pipelines, and control valves are arranged outside the pipelines. The second mixing assembly and the diversion assembly are provided with a connecting plate 6012. A pipeline is communicated with the bottom of the connecting plate 6012, and a control valve is arranged outside the pipeline. The connecting plate 6012 is fixedly connected to the inner wall of the low-temperature freezing tank 601. A turbidity sensor 607 is arranged on the inner wall of the low-temperature freezing tank 601, and the turbidity sensor 607 is arranged between the first diversion plate 6013 and the second diversion plate 6014.

[0050] Specifically, through the low-temperature freezing tank 601, in cooperation with the liquid nitrogen device 602, by storing the enzyme solution and utilizing the low-temperature environment provided by the liquid nitrogen device 602, the freezing treatment of the enzyme solution is achieved, which helps to stabilize the physical and chemical properties of the enzyme solution and prevent the enzyme from deteriorating or inactivating during the treatment process; through the finished enzyme solution collection tank 606, in cooperation with the pipeline and control valve, when it is necessary to collect the qualified enzyme solution, by opening the control valve on the corresponding pipeline, the qualified enzyme solution is introduced into it, realizing the separate collection of high-quality enzyme solution, which is convenient for subsequent use or storage; through the waste liquid collection tank 608, in cooperation with the pipeline and control valve, when detecting the waste liquid that does not meet the requirements, the control valve on the corresponding pipeline is opened, and the waste liquid is introduced into it, realizing the safe storage of the waste liquid and avoiding the waste liquid from contaminating other components or products; through the first spray tank 603, in cooperation with the pipeline and control valve, after opening the corresponding control valve, the weakly polar solvent is sprayed into the enzyme solution in the low-temperature freezing tank 601, realizing the effective removal of non-polar impurities in the enzyme solution by utilizing the physical and chemical properties of the weakly polar solvent and improving the purity of the enzyme solution; through the second spray tank 604, in cooperation with the pipeline and control valve, by opening the corresponding control valve and spraying the strongly polar solvent, and utilizing the interaction between the strongly polar solvent and the polar components in the enzyme solution, the possible residual polar inhibitors in the enzyme solution are removed, ensuring the purity of the enzyme; through the third spray tank 605, in cooperation with the pipeline and control valve, by controlling the opening of the control valve and spraying the inert solvent onto the enzyme solution, and utilizing the properties of the inert solvent, the cleaning and neutralization of the enzyme solution are realized, making the enzyme solution reach the required purity and chemical balance; the turbidity sensor 607 is installed between the first deflector 6013 and the second deflector 6014, and can real-time monitor the purity difference of the liquid after being split by the deflector, so as to accurately judge the states of the waste liquid and the finished enzyme solution. Through the dynamic feedback of the turbidity data, the system can accurately control the outlet valves of the waste liquid and the finished enzyme solution, ensuring that the part of the waste liquid containing high-concentration impurities is discharged in time, while the high-purity finished enzyme solution can smoothly enter the collection tank; the pipeline connected to the bottom of the connecting plate 6012, in cooperation with the connecting plate 6012, serves as the flow channel of the enzyme solution, and is opened or closed under the control of the control valve, realizing the guiding of the treated enzyme solution in the low-temperature freezing tank 601 to the next process.

[0051] Please refer to Figure 9 , a second mixing component is arranged inside the low-temperature freezing tank 601. The second mixing component is used for mixing the enzyme solution inside the low-temperature freezing tank 601. The second mixing component includes a second motor 609, the output end of the second motor 609 is fixedly connected with a second connecting rod 6010, and the bottom of the second connecting rod 6010 is fixedly connected with a second mixing head 6011.

[0052] Specifically, the connecting rod two 6010 and the mixing head two 6011 at its bottom are driven by the motor two 609 to achieve efficient mixing of the enzyme solution in the tank, ensuring uniform distribution and dynamic flow of the enzyme solution in a low-temperature environment, which helps to avoid local supercooling or uneven concentration of the enzyme solution, thus maintaining the stability of the three-dimensional conformation of the enzyme throughout the reaction process, maximizing the activity and stability of the enzyme, enhancing the contact efficiency between the solution and the solvent, further improving the cleaning effect and reaction rate, and laying a foundation for subsequent separation and collection of high-purity finished enzyme solution.

[0053] Please refer to Figure 9 , a diversion component is provided on the inner wall of the low-temperature freezing tank 601. The diversion component is used to divert the enzyme solution. The diversion component includes a diversion plate one 6013 and a diversion plate two 6014. The diversion plate one 6013 is fixedly connected to the inner wall of the outer shell 1, and the diversion plate two 6014 is fixedly connected to the inner wall of the outer shell 1.

[0054] Specifically, through the cooperation of the diversion plate one 6013 and the diversion plate two 6014, the enzyme solution is guided to flow along a preset path inside the low-temperature freezing tank 601, forming a stable diversion effect, and realizing the preliminary physical separation of the finished enzyme solution and the waste liquid.

[0055] Please refer to Figures 3-4 , the pH adjustment mechanism 7 includes a pH controller 701. The pH controller 701 is installed on the inner side wall of the outer shell 1. The pH controller 701 is connected to a pH sensor 702. The detection part of the pH sensor 702 is located inside the serpentine tube 406. An injection pump one 704 and an injection pump two 706 are respectively installed on the inner side wall of the outer shell 1. The input end of the injection pump one 704 is connected to an acid solution storage bottle 703 through a pipeline, and the input end of the injection pump two 706 is connected to an alkali solution storage bottle 705 through a pipeline. The output end of the injection pump one 704 is connected to the serpentine tube 406 through a pipeline, and the output end of the injection pump two 706 is connected to the serpentine tube 406 through a pipeline.

[0056] Specifically, through the cooperation of the pH controller 701 and the pH sensor 702, the pH value of the enzyme solution in the serpentine tube 406 is detected in real time, and the acid-base changes during the reaction process are dynamically obtained, realizing the monitoring of the reaction environment; through the cooperation of the injection pump one 704 and the acid solution storage bottle 703, acid solution is injected as needed according to the instruction of the pH controller 701, quickly adjusting the acidic environment of the enzyme solution, and realizing the effective correction of the over-alkaline reaction; through the cooperation of the injection pump two 706 and the alkali solution storage bottle 705, alkali solution is injected as needed according to the instruction of the pH controller 701, timely adjusting the alkaline environment of the enzyme solution, and realizing the rapid correction of the over-acid reaction; through the overall coordinated action of the above components, it is ensured that the enzyme solution in the serpentine tube 406 is always in the optimal pH range, providing an accurate and stable reaction environment for the efficient binding of the enzyme and the inhibitor, and improving the reaction efficiency and the quality of the finished enzyme solution.

[0057] Please refer to Figure 3 , the reaction combination mechanism 4 further includes a detector 407, which is installed on the inner top wall of the housing 1, and the detection head of the detector 407 is located outside the coiled tube 406.

[0058] Specifically, through the detector 407 and in cooperation with the detection head of the detector 407, the reaction process of the enzyme solution in the coiled tube 406 is monitored in real time. The detector 407 is a UV-Vis detector, which uses non-contact detection technology to accurately collect the key parameters of the reactants and products in the enzyme solution, realizing the control of the reaction process.

[0059] Please refer to Figure 5 , the solvent gradient conversion mechanism 5 further includes a laser Raman spectrometer 506, which is installed on the top of the partition 3, and the detection head of the laser Raman spectrometer 506 is located inside the pipeline connecting the second mixing tank 503 and the low-temperature freezing tank 601.

[0060] Specifically, through the laser Raman spectrometer 506 and in cooperation with the detection head of the laser Raman spectrometer 506, the composition of the solution in the pipeline connecting the second mixing tank 503 and the low-temperature freezing tank 601 is detected in real time. The laser Raman scattering technology is used to perform high-precision analysis on the molecular vibration characteristics in the solution, realizing the efficient monitoring of the binding state of the enzyme solution and the inhibitor during the solvent gradient conversion process.

[0061] Please refer to Figures 1-9 , the present invention also provides a preparation method for bioimprinted enzyme, which includes the following steps:

[0062] S1. Add the enzyme solution to the enzyme solution storage bottle 401, add the inhibitor solution to the inhibitor solution storage bottle 402, start the first feeding pump 403 and the second feeding pump 404, and respectively transport the enzyme solution and the inhibitor solution to the coiled tube 406 of the double-layer constant-temperature reaction box 405 through pipelines to provide a uniformly flowing basic solution for subsequent reactions;

[0063] S2. The enzyme and the inhibitor are fully contacted through the coiled tube 406 in the double-layer constant-temperature reaction box 405, and a binding reaction is carried out under constant-temperature conditions. The pH sensor 702 monitors the pH value of the solution in real time, and the pH controller 701 adjusts the dropping amounts of the acid solution and the alkali solution through the first injection pump 704 and the second injection pump 706 to ensure that the reaction is within the optimal pH range. The binding efficiency is monitored by the detector 407, and after reaching the set requirements, the next step is entered;

[0064] S3. The combined enzyme inhibitor composite solution is successively fed into the first mixing tank 501 and the second mixing tank 503 through the gradient solvent pump 505. The first mixing component completes the full mixing of the solution in the second mixing tank 503. The first cooling jacket 502 and the second cooling jacket 504 maintain a low-temperature environment. The gradient solvent pump 505 gradually adjusts the ratio of the aqueous solution to the lipophilic solvent, and the laser Raman spectrometer 506 conducts detection.

[0065] S4. The solution after solvent gradient conversion enters the low-temperature freezing tank 601 through a pipeline. The liquid nitrogen device 602 maintains the temperature of the low-temperature freezing tank 601 at -10°C. Then, through the first spraying tank 603, a weakly polar solvent is sprayed to remove non-polar impurities in the enzyme solution. Through the second spraying tank 604, a strongly polar solvent is sprayed to remove polar inhibitors on the surface of the enzyme. Through the third spraying tank 605, an inert solvent is sprayed to finally clean and neutralize the enzyme solution to ensure its purity.

[0066] S5. The second mixing component ensures the uniform distribution and dynamic flow of the enzyme solution, so that the three-dimensional conformation of the enzyme is "frozen" in a low-temperature environment, ensuring the high activity and stability of the enzyme.

[0067] S6. The enzyme solution after mixing and spraying is discharged by opening the control valve of the pipeline on the connecting plate 6012. The deflector is inclined to guide the enzyme solution to flow towards the bottom. Since the waste liquid contains a large amount of inhibitor residues and impurities, these components usually have a higher density than the finished enzyme solution. The waste liquid with a higher density is more likely to settle to the bottom of the low-temperature freezing tank 601 under the action of gravity and the first deflector 6013 and the second deflector 6014. When the waste liquid and the finished enzyme solution flow through the diversion area, the turbidity sensor 607 detects the purity of the liquid in real time. If the turbidity is high, it is determined as waste liquid, and the control valve of the pipeline connecting the low-temperature freezing tank 601 to the waste liquid collection tank 608 is opened to discharge it into the waste liquid collection tank 608. If the turbidity is low, it is determined as the finished enzyme solution, and the control valve of the pipeline connecting the low-temperature freezing tank 601 to the finished enzyme solution collection tank 606 is opened. In the finished enzyme solution collection tank 606, the waste liquid and the finished enzyme are controlled to be shunted through independent control valves to avoid mixing or contamination.

[0068] S7. The whole process is automatically managed by the PLC control system. The key parameters such as flow rate, temperature, and pH value are dynamically adjusted through the control panel 2. The system records and feeds back various data, optimizes the reaction conditions in real time, ensures the stable and efficient operation of the device, and provides a basis for subsequent process improvement.

[0069] Specifically, by utilizing the conveying functions of the first feed pump 403 and the second feed pump 404 in the present invention and cooperating with the serpentine tube 406 in the double-layer constant-temperature reaction box 405, it is ensured that the enzyme solution and the inhibitor solution are in full contact and uniformly flow under constant-temperature conditions; the pH value of the reaction environment is synergistically adjusted by the pH sensor 702, the pH controller 701, the first injection pump 704 and the second injection pump 706 to achieve the optimal binding reaction efficiency; the combined enzyme-inhibitor composite solution is fully mixed in the gradient solvent pump 505, the first mixing tank 501 and the second mixing tank 503 through the action of the first mixing component, and the first cooling jacket 502 and the second cooling jacket 504 maintain a low-temperature environment, and the laser Raman spectrometer 506 monitors the reaction state in real time to ensure the accuracy of the solvent gradient conversion; after the solution enters the low-temperature freezing tank 601, the low temperature is maintained by the liquid nitrogen device 602, and the multi-stage cleaning effects of the first spray tank 603, the second spray tank 604 and the third spray tank 605 are coordinated to gradually remove the impurities on the surface of the enzyme and the inhibitor; the second mixing component ensures the uniform distribution and dynamic flow of the enzyme solution in the low-temperature environment, so that the three-dimensional conformation of the enzyme can be stably "frozen" and maintain high activity; through the partition design of the first deflector 6013 and the second deflector 6014 and the real-time monitoring of the turbidity sensor 607, the separation of the finished enzyme solution and the waste liquid is realized, and the shunt of the independent control valve avoids mixing and contamination; the whole process is automatically managed by the PLC control system, the key parameters are dynamically adjusted and the process is optimized in real time, ensuring the high efficiency and stability of the device operation and the high purity of the finished enzyme solution, and at the same time providing a reliable basis for the subsequent process improvement.

[0070] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation device for bioimprinting enzymes, comprising a housing (1), characterized in that, On the outer side of the said housing (1), a control panel (2) is provided. On the inner wall of the housing (1), a partition (3) is fixedly connected. On the top of the housing (1), a reaction combination mechanism (4) is provided. On the top of the partition (3), a solvent gradient conversion mechanism (5) is provided. The reaction combination mechanism (4) is connected to the solvent gradient conversion mechanism (5). On the inner bottom wall of the housing (1), an enzyme solution treatment and recovery mechanism (6) is provided. The solvent gradient conversion mechanism (5) is connected to the enzyme solution treatment and recovery mechanism (6). On the inner side wall of the housing (1), a pH adjustment mechanism (7) is provided. The pH adjustment mechanism (7) is connected to the reaction combination mechanism (4). The said reaction combination mechanism (4) includes an enzyme solution storage bottle (401) and an inhibitor solution storage bottle (402). Both the enzyme solution storage bottle (401) and the inhibitor solution storage bottle (402) are fixedly connected to the top of the housing (1). On the top of the housing (1), a feed pump one (403) and a feed pump two (404) are respectively installed. The input end of the feed pump one (403) is communicated with the enzyme solution storage bottle (401) through a pipeline. The input end of the feed pump two (404) is connected to the inhibitor solution storage bottle (402) through a pipeline. On the inner side wall of the housing (1), a double-layer constant temperature reaction box (405) is fixedly connected. Inside the double-layer constant temperature reaction box (405), a serpentine tube (406) is provided. The output end of the feed pump one (403) is communicated with the serpentine tube (406) through a pipeline. The output end of the feed pump two (404) is communicated with the serpentine tube (406) through a pipeline. A control valve is provided on the outer side of the serpentine tube (406).

2. The preparation device for bioimprinting enzyme according to claim 1, wherein, The said solvent gradient conversion mechanism (5) includes a mixing tank two (503) and a gradient solvent pump (505). The mixing tank two (503) is fixedly connected to the top of the partition (3). On the outer side of the mixing tank two (503), a cooling jacket two (504) is provided. The gradient solvent pump (505) is installed at the bottom of the double-layer constant temperature reaction box (405). The input end of the gradient solvent pump (505) is connected to one end of the serpentine tube (406) passing through the outside of the double-layer constant temperature reaction box (405). The output end of the gradient solvent pump (505) is connected to three mixing tanks one (501) through a pipeline. On the outer side of the mixing tank one (501), a cooling jacket one (502) is provided. The mixing tank one (501) close to the partition (3) side is connected to the mixing tank two (503) through a pipeline, and a control valve is provided on the outer side of the pipeline. Inside the mixing tank two (503), a mixing component one is provided. The mixing component one is used to mix the enzyme solution inside the mixing tank two (503).

3. The preparation device for a bioimprinted enzyme according to claim 2, characterized in that, The said mixing component one includes a motor one (507). The output end of the motor one (507) is fixedly connected to a connecting rod one (508). On the outer side of the connecting rod one (508), a plurality of mixing rods one (5010) are fixedly connected. The bottom of the connecting rod one (508) is fixedly connected to a mixing head one (509).

4. A preparation device for bioimprinting enzyme according to claim 1, characterized in that, The enzyme solution treatment and recovery mechanism (6) includes a low-temperature freezing tank (601), the low-temperature freezing tank (601) is fixedly connected to the inner bottom wall of the outer shell (1), a liquid nitrogen device (602) is arranged outside the low-temperature freezing tank (601), the low-temperature freezing tank (601) is connected to the second mixing tank (503) through a pipeline, and a control valve is arranged outside the pipeline. On one side of the inner bottom wall of the outer shell (1), a finished enzyme solution collection tank (606) is fixedly connected, and on the other side of the inner bottom wall of the outer shell (1), a waste liquid collection tank (608) is fixedly connected. Both the finished enzyme solution collection tank (606) and the waste liquid collection tank (608) are connected to the low-temperature freezing tank (601) through pipelines, and a control valve is arranged outside the pipeline. On the inner bottom wall of the outer shell (1), a first spray tank (603), a second spray tank (604) and a third spray tank (605) are fixedly connected. The first spray tank (603), the second spray tank (604) and the third spray tank (605) are all connected to the low-temperature freezing tank (601) through pipelines, and a control valve is arranged outside the pipeline. A second mixing assembly is arranged inside the low-temperature freezing tank (601), and the second mixing assembly is used for mixing the enzyme solution inside the low-temperature freezing tank (601). A diversion assembly is arranged on the inner wall of the low-temperature freezing tank (601), and the diversion assembly is used for diverting the enzyme solution. A connecting plate (6012) is arranged between the second mixing assembly and the diversion assembly. A pipeline is communicated with the bottom of the connecting plate (6012), and a control valve is arranged outside the pipeline. The connecting plate (6012) is fixedly connected to the inner wall of the low-temperature freezing tank (601). A turbidity sensor (607) is arranged on the inner wall of the low-temperature freezing tank (601).

5. The preparation device for bioimprinting enzyme according to claim 4, characterized in that, The second mixing assembly includes a second motor (609), the output end of the second motor (609) is fixedly connected with a second connecting rod (6010), and the bottom of the second connecting rod (6010) is fixedly connected with a second mixing head (6011).

6. The preparation device for bioimprinting enzyme according to claim 4, characterized in that, The diversion assembly includes a first diversion plate (6013) and a second diversion plate (6014). The first diversion plate (6013) is fixedly connected to the inner wall of the outer shell (1), the second diversion plate (6014) is fixedly connected to the inner wall of the outer shell (1), and the turbidity sensor (607) is arranged between the first diversion plate (6013) and the second diversion plate (6014).

7. The preparation device for bioimprinting enzyme according to claim 1, characterized in that, The pH adjustment mechanism (7) includes a pH controller (701) installed on the inner side wall of the housing (1). The pH controller (701) is connected to a pH sensor (702), and the detection part of the pH sensor (702) is located inside the serpentine tube (406). On the inner side wall of the housing (1), an injection pump one (704) and an injection pump two (706) are respectively installed. The input end of the injection pump one (704) is communicated with an acid solution storage bottle (703) through a pipeline, and the input end of the injection pump two (706) is communicated with an alkali solution storage bottle (705) through a pipeline. The output end of the injection pump one (704) is communicated with the serpentine tube (406) through a pipeline, and the output end of the injection pump two (706) is communicated with the serpentine tube (406) through a pipeline.

8. A preparation device for bioimprinting enzyme according to claim 1, characterized in that, The reaction combination mechanism (4) further includes a detector (407) installed on the inner top wall of the housing (1), and the detection head of the detector (407) is located outside the serpentine tube (406).

9. The preparation device for a bioimprinted enzyme according to claim 1, characterized in that, The solvent gradient conversion mechanism (5) further includes a laser Raman spectrometer (506) installed on the top of the partition plate (3), and the detection head of the laser Raman spectrometer (506) is located inside the pipeline connecting the second mixing tank (503) and the low-temperature freezing tank (601).

10. A preparation method for a bioimprinted enzyme, according to the preparation device for a bioimprinted enzyme described in any one of claims 1-9, characterized in that, It includes the following steps: S1. Add the enzyme solution to the enzyme solution storage bottle (401), add the inhibitor solution to the inhibitor solution storage bottle (402), start the feeding pump one (403) and the feeding pump two (404), and respectively transport the enzyme solution and the inhibitor solution to the serpentine tube (406) of the double-layer constant-temperature reaction box (405) through pipelines, providing a uniformly flowing basic solution for subsequent reactions. S2. The enzyme and the inhibitor fully contact through the serpentine tube (406) in the double-layer constant-temperature reaction box (405) and carry out a binding reaction under constant-temperature conditions. The pH sensor (702) monitors the pH value of the solution in real time, and the pH controller (701) adjusts the dropping amounts of the acid solution and the alkali solution through the injection pump one (704) and the injection pump two (706) to ensure that the reaction is within the optimal pH range. The binding efficiency is monitored by the detector (407), and after reaching the set requirements, proceed to the next step. S3. The combined enzyme-inhibitor composite solution is successively fed into the three first mixing tanks (501) and the second mixing tank (503) through the gradient solvent pump (505). The first mixing component completes the full mixing of the solution in the second mixing tank (503). The first cooling jacket (502) and the second cooling jacket (504) maintain a low-temperature environment. The gradient solvent pump (505) gradually adjusts the ratio of the aqueous solution to the lipophilic solvent, and the laser Raman spectrometer (506) conducts detections. S4. The solution after solvent gradient conversion enters the low-temperature freezing tank (601) through a pipeline. The liquid nitrogen device (602) maintains the temperature of the low-temperature freezing tank (601) at -10°C. Then, it passes through the first spraying tank (603) to spray a weakly polar solvent to remove non-polar impurities in the enzyme solution, the second spraying tank (604) to spray a strongly polar solvent to remove polar inhibitors on the surface of the enzyme, and the third spraying tank (605) to spray an inert solvent to finally clean and neutralize the enzyme solution and ensure its purity. S5. The second mixing component ensures the uniform distribution and dynamic flow of the enzyme solution, "freezing" the three-dimensional conformation of the enzyme in a low-temperature environment to ensure the high activity and stability of the enzyme. S6. The enzyme solution that has been mixed and sprayed is discharged by opening the control valve of the pipeline on the connecting plate (6012). The deflector is inclined to guide the enzyme solution to flow towards the bottom. Since the waste liquid contains a large amount of inhibitor residues and impurities, these components usually have a higher density than the finished enzyme solution. The waste liquid with a higher density is more likely to settle to the bottom of the low-temperature freezing tank (601) under the action of gravity and the first deflector (6013) and the second deflector (6014). When the waste liquid and the finished enzyme solution flow through the diversion area, the turbidity sensor (607) detects the purity of the liquid in real time. If the turbidity is high, it is determined as waste liquid, and the control valve on the pipeline connecting the low-temperature freezing tank (601) to the waste liquid collection tank (608) is opened to discharge it into the waste liquid collection tank (608). If the turbidity is low, it is determined as the finished enzyme solution, and the control valve on the pipeline connecting the low-temperature freezing tank (601) to the finished enzyme solution collection tank (606) is opened. In the finished enzyme solution collection tank (606), the waste liquid and the finished enzyme are controlled to be separated by independent control valves to avoid mixing or contamination. S7. The entire process is automatically managed by the PLC control system. Key parameters such as flow rate, temperature, and pH value are dynamically adjusted through the control panel (2). The system records and feeds back various data, optimizes the reaction conditions in real time to ensure the stable and efficient operation of the device, and provides a basis for subsequent process improvement.

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