A lactate dehydrogenase purification system

Abstract

This invention discloses a lactate dehydrogenase purification system, comprising: a filtration and pre-cooling device for filtering and cooling a crude enzyme solution containing lactate dehydrogenase; a storage system including multiple buffer tanks for storing buffer solutions, and a sample tank connected to the outlet of the filtration and pre-cooling device for collecting the filtered and cooled enzyme solution; a delivery system for sequentially pumping out the enzyme solution from the sample tank and the buffer solution from the buffer tank; a purification device for purifying the enzyme solution using the buffer solution; a collection system for systematically collecting the liquid discharged from different stages by the purification device; and a monitoring system including a conductivity meter, a pH meter, an ultraviolet detector, and a thermometer, sequentially installed along the inlet direction on the pipeline between the outlet of the purification device and the inlet of the collection system. By pre-cooling the crude enzyme solution containing lactate dehydrogenase and performing real-time monitoring during the purification process, continuous purification of lactate dehydrogenase can be achieved.

Description

Technical Field

[0001] This utility model relates to a lactate dehydrogenase purification system, belonging to the field of lactate dehydrogenase purification technology. Background Technology

[0002] In the preparation of lactate dehydrogenase, recombinant plasmids containing the lactate dehydrogenase gene are transferred into *E. coli* to obtain *E. coli* cells expressing lactate dehydrogenase. The next step is to purify the lactate dehydrogenase protein. Currently, the practically feasible method for purifying lactate dehydrogenase is high-power sonication. After complete disruption, the supernatant is collected by centrifugation and purified using ion-exchange chromatography. However, this method does not pre-treat the sample before purification. Excessive sample temperature before purification can affect the purification effect. Furthermore, the purification process cannot monitor experimental data in real time, compromising experimental continuity and hindering the timely execution of subsequent experiments. Therefore, establishing a good lactate dehydrogenase purification system can reduce or avoid certain problems during purification, thus improving the purification efficiency. Utility Model Content

[0003] The purpose of this invention is to provide a lactate dehydrogenase purification system that enables continuous purification of lactate dehydrogenase by pre-cooling the crude enzyme solution containing lactate dehydrogenase and monitoring it in real time during the purification process.

[0004] To achieve the above objectives, the technical solution adopted by this utility model is as follows: A lactate dehydrogenase purification system, comprising: A filtration and precooling device is used to filter and cool the crude enzyme solution containing lactate dehydrogenase. The liquid storage system includes multiple buffer tanks for storing buffer solutions, and a sample tank connected to the outlet of the filtration precooling device for collecting the filtered and cooled enzyme solution. The liquid delivery system is connected to the sample container and multiple buffer tanks, and is used to pump out the enzyme solution in the sample container and the buffer solution in the buffer tanks in sequence. The purification device is connected to the outlet of the liquid delivery system and is used to purify the enzyme solution using a buffer solution. The liquid collection system is connected to the liquid outlet of the purification device and is used to collect liquids from different stages discharged from the purification device in an orderly manner. The monitoring system includes a conductivity meter, a pH meter, an ultraviolet detector, and a thermometer, which are installed sequentially along the liquid inlet direction on the pipeline between the liquid outlet of the purification device and the liquid inlet of the collection system.

[0005] Preferably, the filtration and precooling device includes a hopper, a filter, a condenser, and a first peristaltic pump connected in sequence along the liquid inlet direction, and the liquid outlet of the first peristaltic pump is connected to the sample container through a pipe.

[0006] Preferably, a filter cloth is also provided inside the hopper.

[0007] Preferably, the filter includes a transparent filter tube, and a filter element is disposed inside the rear tube of the filter tube; Both ends of the filter tube are detachably connected to the pipes used to connect the funnel and the condenser tube, and a drain pipe with a valve is provided below the filter tube located in front of the filter element.

[0008] Preferably, the condenser is a shell-and-tube condenser, and both ends are detachably connected to the pipes used to connect the filter and the first peristaltic pump.

[0009] Preferably, the liquid delivery system includes a first multi-channel proportional valve, a second peristaltic pump, and a mixer (static baffle type or orifice plate type); wherein, The multiple inlet terminals of the first multi-channel proportional valve are respectively connected to the sample tank and multiple buffer tanks. The inlet of the second peristaltic pump is connected to the outlet of the first multi-channel proportional valve, and the outlet is connected to the inlet of the mixer. The outlet of the mixer is connected to the inlet of the purification device.

[0010] Preferably, each buffer tank includes a tank body and a cover on top of the tank body. An inlet tube, a replenishment tube, and an exhaust tube are provided on the cover. The inlet tube extends to the bottom of the tank body, and the end of the inlet tube forms a conical structure with an inlet head having an inlet hole. The top of the inlet tube is connected to the corresponding liquid inlet end on the first multi-channel proportional valve through a pipe.

[0011] Preferably, the purification device is an anion exchange chromatography column.

[0012] Preferably, the liquid collection system includes a second multi-channel proportional valve and multiple liquid collection tanks. The inlet end of the second multi-channel proportional valve is connected to the outlet end of the purification device, and the multiple outlet ends of the second multi-channel proportional valve are respectively connected to the multiple liquid collection tanks.

[0013] The beneficial effects of this utility model are as follows: To address the issues of excessively high temperatures affecting lactate dehydrogenase purification, experimental discontinuity, and the inability to monitor experimental data in real time during experiments, the purification method provided in this application utilizes a pre-cooling filter to cool the crude enzyme solution and employs a monitoring system for real-time monitoring. A multi-channel proportional valve is used to adjust the buffer composition, resulting in a simple operation and enabling continuous purification of lactate dehydrogenase.

[0014] Compared with existing methods, this system can achieve efficient and stable purification of lactate dehydrogenase. The filtration and precooling device ensures sample clarity and improves sample stability. By integrating five functions, namely filtration, liquid injection, purification, real-time monitoring and protein collection, lactate dehydrogenase can be purified completely and efficiently. Attached Figure Description

[0015] Figure 1 is a structural diagram of the purification system; Figure 2 is a structural diagram of the filter precooling device; Figure 3 is a structural diagram of the liquid storage system and the liquid delivery system; Figure 4 is a structural diagram of the purification device; Figure 5 is a structural diagram of the monitoring system and the liquid collection system.

[0016] The meanings of the main reference numerals in the figure are as follows: 1. Filtration and precooling device; 2. Liquid storage system; 3. Liquid delivery system; 4. Purification device; 5. Liquid collection system; 6. Monitoring system; 7. Sample container; 8. Conductivity meter; 9. pH meter; 10. UV detector; 11. Thermometer; 12. Hopper; 13. Filter; 14. Condenser; 15. First peristaltic pump; 16. Filter cloth; 17. Filter tube; 18. Wood fiber filter element; 19. Discharge pipe; 20. Valve; 21. First multi-channel proportional valve; 22. Second peristaltic pump; 23. Mixer; 24. Container body; 25. Cover; 26. Sample inlet tube; 27. Sample replenishment tube; 28. Exhaust pipe; 29. ​​Sample inlet head; 30. Equilibration buffer container; 31. Elution buffer container; 32. Column cleaning buffer container; 33. Column preservation buffer container; 34. Second multi-channel proportional valve; 35. Liquid collection container. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0018] This embodiment provides a lactate dehydrogenase purification system, such as Figure 1-5 As shown, the system includes a filtration pre-cooling device 1, a storage system 2, a delivery system 3, a purification device 4, a collection system 5, and a monitoring system 6. The filtration pre-cooling device 1 is used to filter and cool the crude enzyme solution containing lactate dehydrogenase. The storage system 2 includes multiple buffer tanks for storing buffer solutions, and a sample tank 7 connected to the outlet of the filtration pre-cooling device 1 for collecting the filtered and cooled enzyme solution.

[0019] The delivery system 3 is connected to the sample container 7 and multiple buffer containers, and is used to sequentially pump out the enzyme solution in the sample container 7 and the buffer solution in the buffer containers. The purification device 4 is connected to the outlet of the delivery system 3, and is used to purify the enzyme solution using buffer solution. The collection system 5 is connected to the outlet of the purification device 4, and is used to systematically collect the liquid discharged from different stages by the purification device 4. The monitoring system 6 includes a conductivity meter 8, a pH meter 9, an ultraviolet detector 10, and a thermometer 11, which are sequentially installed along the inlet direction on the pipeline between the outlet of the purification device 4 and the inlet of the collection system 5.

[0020] Specifically, the filtration and precooling device 1 includes a hopper 12, a filter 13, a condenser 14, and a first peristaltic pump 15 connected sequentially along the liquid inlet direction. The hopper 12 has a conical structure, and a 400-mesh filter cloth 16 is placed inside the hopper 12. The filter cloth 16 is folded into a cone shape and placed inside the hopper 12 and adheres to the inner wall of the hopper 12. The filter cloth 16 has a certain filtering effect on the supernatant of the bacterial cells after centrifugation.

[0021] The filter 13 includes a transparent filter tube 17, with a wood fiber filter element 18 inserted into the rear part of the tube. After passing through the wood fiber filter element 18, the sample achieves good clarity, removing suspended solids and particulate matter from the supernatant. Both ends of the filter tube 17 are internally threaded, connecting to a pipe used for connecting the funnel and condenser via these threads. A drain pipe 19 with a valve 20 is located below the filter tube 17, in front of the wood fiber filter element 18. The transparent filter tube 17 allows for easy observation of any blockages, facilitating timely opening of the valve 20. If blockages or impaired liquid flow are observed within the filter tube 17 during filtration, the valve 20 on the drain pipe can be opened to expel the blockage from the filter tube 17.

[0022] The condenser 14 is a transparent shell-and-tube condenser (crude enzyme solution flows through the tube side, cooling water flows through the shell side), and both ends are detachably connected (threaded) to the pipes connecting the filter 13 and the first peristaltic pump 15. In practical applications, a thermometer 10 is also installed on the pipe between the condenser 14 and the first peristaltic pump 15. By controlling the flow rate of cooling water in the shell side, pre-cooling of the crude enzyme solution (2-8℃) can be achieved, which can improve protein stability. The outlet end of the first peristaltic pump 15 is connected to the sample container 7 through a pipe.

[0023] The liquid delivery system 3 includes a first multi-channel proportional valve 21, a second peristaltic pump 22, and a mixer 23; wherein, multiple inlet terminals of the first multi-channel proportional valve 21 are respectively connected to the sample tank 7 and multiple buffer tanks; the inlet terminal of the second peristaltic pump 22 is connected to the outlet terminal of the first multi-channel proportional valve 21, and the outlet terminal is connected to the inlet terminal of the mixer 23; the outlet terminal of the mixer 23 is connected to the inlet terminal of the purification device 4.

[0024] Each buffer tank includes a tank body 24 and a cover 25 covering the top of the tank body 24. The cover 25 is equipped with an inlet tube 26, a replenishment tube 27, and an exhaust tube 28 (equipped with an exhaust valve). In practical applications, the sample tank 7 is also equipped with an exhaust tube 28 with an exhaust valve. The inlet tube 26 extends to the bottom of the tank body 24, and its end forms a tapered inlet head 29 with an inlet orifice. In practical applications, a filter cloth 16 is wrapped around the inlet head 29 and secured to it using a clamp or other fasteners. The top of the inlet tube 26 is connected to the corresponding inlet end of the first multi-channel proportional valve 21 via a pipe.

[0025] Specifically, the buffer tanks include an equilibration buffer tank 30, an elution buffer tank 31, a column cleaning buffer tank 32, and a column preservation buffer tank 33. Five infusion tubes are connected to the inlet of the first multi-channel proportional valve 21. The first infusion tube is connected to the inlet tube 26 of the sample tank 7; the second infusion tube is connected to the inlet tube 26 of the equilibration buffer tank 30; the third infusion tube is connected to the inlet tube 26 of the elution buffer tank 31; the fourth infusion tube is connected to the inlet tube 26 of the column cleaning buffer tank 32; and the fifth infusion tube is connected to the inlet tube 26 of the column preservation buffer tank 33.

[0026] The equilibration buffer consisted of 20 mM Tris (pH 8.0) + 150 mM NaCl, the elution buffer consisted of 20 mM Tris (pH 8.0) + 250 mM NaCl, the column washing buffer consisted of 20 mM Tris (pH 8.0) + 1.5 M NaCl, and the column preservation buffer consisted of 20% ethanol.

[0027] Purification device 4 is an anion exchange chromatography column, specifically a Q-HP chromatography column (a strong anion exchange chromatography column manufactured by Cytiva). The collection system 5 includes a second multi-channel proportional valve 34 and multiple collection tanks 35. The inlet of the second multi-channel proportional valve 34 is connected to the outlet of the purification device 4, and the multiple outlets of the second multi-channel proportional valve 34 are respectively connected to the multiple collection tanks 35.

[0028] The multi-channel proportional valve used is the Outlet Valve V9H-O model valve from Beijing Lingmei Sicheng Co., Ltd.

[0029] The purification method is as follows: 1) Weigh the frozen E. coli cells according to the ratio of cell weight to Q-HP packing volume = 2:5, add the cells to the beaker, and add resuspension solution according to the ratio of cell weight to resuspension volume = 1:10 to dissolve them.

[0030] 2) Place the beaker containing the resuspended bacterial cells into ice water, set the ultrasonic power to 650W, turn on for 3 seconds and stop for 4 seconds, and sonicate for 20 minutes until the bacterial solution is relatively clear (if it is not clear, you can extend the sonication time).

[0031] 3) Take a centrifuge tube, add the sonicated bacterial cells, balance the mixture, and centrifuge at 13000 rpm for 30 minutes. Take the supernatant after centrifugation.

[0032] 4) In the pre-cooling filter 1, pour the centrifuged bacterial supernatant into the hopper 12, open the exhaust valve of the sample container 7, and the bacterial supernatant flows through the wood fiber filter element 18 under the action of the first peristaltic pump 15, and then flows into the sample container 7 through the condenser 14 until filtration and cooling are completed.

[0033] 5) If blockage or poor liquid flow is observed in the filter tube 17, open the valve 20 on the discharge pipe 19 connected to the corresponding filter tube 17 to discharge the liquid.

[0034] 6) Switch the first multi-channel proportional valve 21 to the second infusion tube, adjust the second peristaltic pump 22 to the appropriate flow rate range, and draw equilibration buffer through the second peristaltic pump 22 to equilibrate 3-5 column volumes with equilibration buffer.

[0035] 7) Switch the first multi-channel proportional valve 21 to the first infusion tube, adjust the second peristaltic pump 22 to the appropriate flow rate range, and use the second peristaltic pump 22 to draw the bacterial supernatant after passing through the filtration and pre-cooling device 1 into the chromatography column. After the sample loading is completed, switch the first multi-channel proportional valve 21 to the second infusion tube, and drive the second peristaltic pump 22 to flow the equilibration buffer through the Q-HP chromatography column; when the readings of the conductivity meter 8, pH meter 9, and UV detector 10 reach stability, switch the first multi-channel proportional valve 21 to the third infusion tube, and use the elution buffer to dissociate the target protein; after dissociation is completed, switch the first multi-channel proportional valve 21 to the fourth infusion tube, and use the column washing buffer to wash the chromatography column; after washing, switch the first multi-channel proportional valve 21 to the fifth infusion tube, and use the column preservation solution to preserve the column.

[0036] 8) During the elution process, wait for the UV detector to reach 10UV. 280nm Protein collection begins when the UV value rises to 0.2-0.3 Au, and when UV... 280nm Protein collection stops when the value drops to 0.2-0.3 Au; this process is achieved by switching the liquid outlet of the second multi-channel proportional valve 34 to different collection tanks 35 (a label is attached to the collection tank 35 to prevent collection errors).

[0037] 9) Processing the target protein: Dissociate the sample and dialyze it into dialysis buffer. Dialyze three times. After dialysis, concentrate the sample using an ultrafiltration tube or membrane pack until the protein concentration is 5 mg / ml. Add preservative P300 for storage.

Claims

1. A lactate dehydrogenase purification system, characterized in that, include: A filtration and precooling device is used to filter and cool the crude enzyme solution containing lactate dehydrogenase. The liquid storage system includes multiple buffer tanks for storing buffer solutions, and a sample tank connected to the outlet of the filtration precooling device for collecting the filtered and cooled enzyme solution. The liquid delivery system is connected to the sample container and multiple buffer tanks, and is used to pump out the enzyme solution in the sample container and the buffer solution in the buffer tanks in sequence. The purification device is connected to the outlet of the liquid delivery system and is used to purify the enzyme solution using a buffer solution. The liquid collection system is connected to the liquid outlet of the purification device and is used to collect liquids from different stages discharged from the purification device in an orderly manner. The monitoring system includes a conductivity meter, a pH meter, an ultraviolet detector, and a thermometer, which are installed sequentially along the liquid inlet direction on the pipeline between the liquid outlet of the purification device and the liquid inlet of the collection system.

2. The lactate dehydrogenase purification system according to claim 1, characterized in that, The filtration and precooling device includes a hopper, a filter, a condenser, and a first peristaltic pump connected in sequence along the liquid inlet direction. The liquid outlet of the first peristaltic pump is connected to the sample container through a pipe.

3. The lactate dehydrogenase purification system according to claim 2, characterized in that, The hopper is also equipped with filter cloth.

4. The lactate dehydrogenase purification system according to claim 2, characterized in that, The filter includes a transparent filter tube, and a filter element is installed inside the rear part of the filter tube. Both ends of the filter tube are detachably connected to the pipes used to connect the funnel and the condenser tube, and a drain pipe with a valve is provided below the filter tube located in front of the filter element.

5. The lactate dehydrogenase purification system according to claim 4, characterized in that, The condenser is a shell-and-tube condenser, and both ends are detachably connected to the pipes used to connect the filter and the first peristaltic pump.

6. The lactate dehydrogenase purification system according to claim 1, characterized in that, The liquid delivery system includes a first multi-channel proportional valve, a second peristaltic pump, and a mixer; wherein, The multiple inlet terminals of the first multi-channel proportional valve are respectively connected to the sample tank and multiple buffer tanks. The inlet of the second peristaltic pump is connected to the outlet of the first multi-channel proportional valve, and the outlet is connected to the inlet of the mixer. The outlet of the mixer is connected to the inlet of the purification device.

7. The lactate dehydrogenase purification system according to claim 6, characterized in that, Each buffer tank includes a tank body and a cover on top of the tank body. The cover is equipped with an injection tube, a replenishment tube, and an exhaust tube. The injection tube extends to the bottom of the tank body, and the end of the injection tube forms a conical structure with an injection head having an injection hole. The top of the injection tube is connected to the corresponding liquid inlet end on the first multi-channel proportional valve through a pipe.

8. The lactate dehydrogenase purification system according to claim 1, characterized in that, The purification device is an anion exchange chromatography column.

9. The lactate dehydrogenase purification system according to claim 1, characterized in that, The liquid collection system includes a second multi-channel proportional valve and multiple liquid collection tanks. The inlet end of the second multi-channel proportional valve is connected to the outlet end of the purification device, and the multiple outlet ends of the second multi-channel proportional valve are respectively connected to multiple liquid collection tanks.

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