Device for coupling foam separation in fermentation tank and application of device in production and separation of recombinant protein

Abstract

The invention provides a device for coupling foam separation in a fermentation tank and application of the device to production and separation of recombinant protein, and belongs to the technical field of bioengineering and enzyme engineering. According to the device for coupling foam separation in the fermentation tank, the micron-sized aeration disc is arranged, seed liquid is inoculated into the device for semi-continuous fed-batch fermentation, part of fermentation liquid is taken out every 96 h, a fresh culture medium is supplemented, the influence of metabolite accumulation on the fermentation progress in the fermentation process is effectively solved, product inhibition is relieved, and the production efficiency is improved. The processes of sterilization, cleaning and the like are reduced; after fermentation is finished, a micron-sized aeration disc is used for directly carrying out foam separation on fermentation liquid in the fermentation tank, and efficient separation and concentration of the target recombinant protein are achieved; the device has the advantages of being simple in structure, easy, convenient and rapid to operate, low in time and material cost in the production process, capable of effectively improving the production efficiency and the like, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical fields of bioengineering and enzyme engineering, and particularly relates to a device for coupling foam separation inside a fermentation tank and application thereof in producing and separating recombinant proteins. Background Art

[0002] The Pichia pastoris expression system is a well-established engineered bacterial production system for producing exogenous proteins. To date, thousands of proteins have been expressed in this system. In the food industry, successful commercialization and efficient expression of zearalenone hydrolase, recombinant human-like collagen, and bovine lactoferrin antibacterial peptides have been achieved. Pichia pastoris has been widely used in areas such as improving food safety and producing high-value proteins. Therefore, research on simple, low-cost, and easy-to-operate industrial fermentation processes is of great significance.

[0003] Current Pichia pastoris expression systems for producing exogenous proteins mostly use a fed-batch fermentation method, which consumes significant time and material costs. The fermentation process requires sterilization, seed solution preparation, inoculation, feeding, and cleaning. The separation process often uses methods such as ultrafiltration concentration, ammonium sulfate precipitation, and affinity chromatography. These processes are complex, time-consuming, and have low production efficiency. Therefore, it is necessary to design a fermentation and separation method that addresses the existing issues of high time and material costs, complex equipment and operations, and difficulty in scalable production of recombinant proteins, enabling efficient fermentation and separation of recombinant proteins. Summary of the Invention

[0004] In response to some deficiencies in the prior art, the present invention provides a device for coupling foam separation inside a fermenter and its application in producing and separating recombinant proteins; the device for coupling foam separation inside a fermenter according to the present invention is provided with a micron-level aeration disk, seed liquid is inoculated into the device for semi-continuous fed-batch fermentation, part of the fermentation liquid is taken out every 96 hours, and fresh culture medium is replenished, which effectively improves the negative impact of the accumulation of metabolites on the fermentation progress during the fermentation process, alleviates product inhibition, and reduces processes such as sterilization and cleaning; after the fermentation is completed, the fermentation liquid is subjected to foam separation inside the fermenter using a micron-level aeration disk, thereby achieving efficient separation and concentration of the target recombinant protein; the present invention has the advantages of simple device structure, simple and quick method operation, low time and material costs in the production process, and improved production efficiency, and has good practicality.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention first provides a device for coupled foam separation inside a fermenter, the device comprising: a fermenter body, and a fermenter cover and a fermenter bottom plate respectively connected to the fermenter body;

[0007] The fermentation tank cover is provided with a stirring shaft with stirring blades on one side in contact with the fermentation tank body, and a motor for driving the stirring shaft and stirring blades to rotate on the other side; the fermentation tank cover is provided with multiple electrode interfaces; the fermentation tank cover is also provided with a sample outlet, a sample inlet, a sampling port, an exhaust port, an air inlet and a feed port;

[0008] A stainless steel heating jacket is provided under the fermentation tank body, and the stainless steel heating jacket is connected to a water pipe;

[0009] The fermentation tank chassis comprises a chassis tray supporting the bottom of the fermentation tank and a heating chassis located above the chassis tray; the chassis tray is provided with a port connected to an external control device.

[0010] Preferably, the fermentation tank body is connected to the fermentation tank cover through threads; the fermentation tank body is connected to corresponding concave ports on the chassis tray through a plurality of convex interfaces at the bottom of the fermentation tank body.

[0011] Preferably, the electrode interface is arranged around the motor, and the electrode interface includes a dissolved oxygen electrode interface, a pH electrode interface, a temperature electrode interface and a defoaming electrode interface; the dissolved oxygen electrode interface, the pH electrode interface, the temperature electrode interface and the defoaming electrode interface are respectively connected to the dissolved oxygen electrode, the pH electrode, the temperature electrode and the defoaming electrode.

[0012] Preferably, a condenser connected to the exhaust port is further installed above the fermentation tank cover; the condenser includes a condenser water inlet pipe, a condenser water outlet pipe and an exhaust pipe.

[0013] Preferably, during the foam separation process, the aeration disk is placed inside the fermenter coupled with the foam separation device and is connected to the feed port via a vent pipe;

[0014] The device for coupling foam separation inside the fermentation tank is further provided with a magnetic stirrer and a collecting bottle placed above the magnetic stirrer, and the collecting bottle is connected to the exhaust pipe of the condenser through a silicone tube.

[0015] The present invention also provides the use of the above-mentioned fermentation tank internal coupled foam separation device in the production and separation of recombinant proteins.

[0016] Preferably, the recombinant protein comprises β-glucosidase.

[0017] The present invention also provides a method for producing and isolating a recombinant protein, the method comprising:

[0018] (1) Production of recombinant proteins:

[0019] The seed liquid of the genetically engineered bacteria expressing the recombinant protein is inoculated into the fermentation tank of the fermentation-coupled foam separation device, air is introduced into the device through the air inlet, the air intake is adjusted through the air inlet during the fermentation process, a carbon source is supplemented through the feed port, the state of the fermentation liquid during the fermentation stage is detected by a dissolved oxygen electrode, a pH electrode, and a temperature electrode, and the recombinant protein is produced by batch fermentation using a semi-continuous batch fermentation method to obtain a fermentation liquid;

[0020] (2) Isolation of recombinant protein:

[0021] A collecting bottle is installed on the device coupled with foam separation inside the fermenter and the collecting bottle is placed on a magnetic stirrer. A feeding port is connected to an external gas device. An aeration disk is placed inside the fermenter of the fermenter-coupled foam separation device and connected to the feeding port via a vent pipe. Fermentation liquid enters the device through an inlet, the inlet is closed, gas is blown into the device, foam is collected, and the magnetic stirrer defoams the collected foam. The device realizes foam separation directly inside the fermenter to obtain separated and concentrated recombinant protein.

[0022] Preferably, in step (1), the parameters of the fermentation stage are set as follows: dissolved oxygen 30%, temperature 28° C., and pH 6;

[0023] The carbon source is glycerol before the OD600 value of the bacterial solution reaches 100, and methanol and sorbitol thereafter;

[0024] During the semi-continuous batch fermentation, fermentation samples were collected and supplemented with BSM medium without glycerol; the BSM medium contained: 14.9 g of MgSO4·7H2O, 4.13 g of KOH, 0.93 g of CaSO4·2H2O, 18.2 g of K2SO4, 10 g of (NH4)2SO4, and 26.7 mL of H3PO4, and the volume was adjusted to 1 L with ddH2O.

[0025] Preferably, in step (2), the aeration rate during the foam separation process is controlled at the lowest range of the regulating valve of the external air inlet device, 1 vvm;

[0026] The surfactant Tween 80 is added into the fermentation broth.

[0027] Preferably, the protein concentration in the fermentation broth is 0.05-0.15 mg / mL; the final concentration of the surfactant Tween 80 is 0.4-1.5 mg / mL;

[0028] The temperature of the fermentation liquid after gas injection is 20-40°C.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention designs a method for separating β-glucosidase expressed in Pichia pastoris by coupling foam separation inside a fermenter. The fermentation broth after 96 hours of fermentation is used as seed liquid for semi-continuous fed-batch fermentation. Part of the fermentation broth is removed every 96 hours and supplemented with fresh culture medium. This effectively solves the problem of the impact of metabolite accumulation on the fermentation progress during the fermentation process, alleviates product inhibition, and reduces processes such as sterilization and cleaning.

[0031] After fermentation, the present invention inserts a micron-sized aeration disk into the fermenter to achieve foam separation within the fermenter. The small pore size of the aeration disk, combined with a surfactant, enhances the foaming effect, thereby achieving separation of the target protein. Furthermore, the air intake device of the present invention is relatively independent, and the pore size of the air inlet in the fermenter during fermentation is larger than that of the aeration disk, which helps to suppress excessive foam generation during induced expression.

[0032] The production and separation and concentration methods of the present invention are simple, quick, and low-cost, improving production efficiency. The present invention achieves semi-continuous batch fermentation of recombinant β-glucosidase in Pichia pastoris and couples fermentation with foam separation to achieve concentrated separation of the recombinant protein, saving fermentation time and costs. Simultaneously, the foam separation method is coupled within the fermentor for concentrated separation, effectively simplifying the recombinant protein separation process. While achieving a separation efficiency of 70%, the work intensity and workload are greatly reduced. The present invention has a simple process, is cost-effective, and is suitable for industrial large-scale production of exogenous proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 2 shows a fermentation-coupled foam separation device, Figure A is a schematic diagram of the entire device, Figure B is a top view of the fermentation tank cover, Figure C is a main view of the fermentation tank body, and Figure D is a top view of the fermentation chassis.

[0034] Figure 2 Shows an enlarged view of the condenser (A) and aeration plate (B) in the fermenter.

[0035] Figure 3 Figure 3 shows a comparison of the separation efficiency of fermentation-coupled foam separation under different fermentation broth conditions. Figures A, B, and C show the enrichment ratio, recovery rate, and enzyme activity recovery rate of foam separation under different initial protein concentrations, surfactant concentrations, and temperatures, respectively.

[0036] Figure 4 Shows the fermentation results of different batches of semi-continuous batch fermentation.

[0037] Reference numerals:

[0038] 1-magnetic stirrer; 2-collecting bottle; 3-fermenter body; 4-fermenter cover; 5-condenser; 6-motor; 7-stirring shaft; 8-stirring blade; 9-aeration plate; 10-heating jacket; 11-fermenter chassis; 12-sampling port; 13-sample outlet; 14-exhaust port; 15-air inlet; 16-pH electrode interface; 17-defoaming electrode interface; 18-temperature electrode interface; 19-dissolved oxygen electrode interface; 20-inlet; 21-feeding port; 22-water pipe; 23-convex interface; 24-chassis tray; 25-heating chassis; 26-concave port; 27-port. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0040] Example 1:

[0041] like Figure 1 and 2 As shown, the fermentation tank internal coupled foam separation device of the present invention includes: a fermentation tank body 3, and a fermentation tank cover 4 and a fermentation tank bottom plate 11 respectively connected to the fermentation tank body 3; the fermentation tank cover 4 is provided with a stirring shaft 7 with stirring blades 8 on one side in contact with the fermentation tank body 3, and a motor 6 for driving the stirring shaft 7 and the stirring blades 8 to rotate on the other side; the fermentation tank cover 4 is provided with multiple electrode interfaces; the fermentation tank cover 4 is also provided with a sample outlet 13, a sample inlet 20, a sampling port 12, an exhaust port 14, an air inlet 15 and a feed port 21;

[0042] A stainless steel heating jacket 10 is provided under the fermentation tank body 3, and the stainless steel heating jacket 10 is connected to a water pipe 22; the fermentation tank chassis 11 includes a chassis tray 24 supporting the bottom of the fermentation tank and a heating chassis 25 located above the chassis tray; the chassis tray 24 is provided with a port 27 for connecting to an external control device.

[0043] During implementation, the fermentation tank body 3 and the fermentation tank lid 4 are connected via threads. The fermentation tank body 3 is connected to corresponding recessed ports 26 on the chassis tray 24 via multiple male ports 23 on the bottom of the fermentation tank body 3. Electrode ports are located around the motor 6 and include a dissolved oxygen electrode port 19, a pH electrode port 16, a temperature electrode port 18, and a defoaming electrode port 17. The dissolved oxygen electrode port 19, pH electrode port 16, temperature electrode port 18, and defoaming electrode port 17 are connected to the dissolved oxygen electrode, pH electrode, temperature electrode, and defoaming electrode, respectively. A condenser 5 connected to the exhaust port 14 is also installed above the fermentation tank lid 4. The condenser 5 includes a condenser water inlet pipe, a condenser water outlet pipe, and an exhaust pipe.

[0044] During the specific implementation process, when fermentation separation is performed using the fermentation tank internally coupled foam separation device, the aeration plate 9 is placed inside the fermentation tank of the fermentation tank coupled foam separation device, and the aeration plate 9 is connected to the feed port 21 via a vent pipe. The fermentation tank internally coupled foam separation device is also provided with a magnetic stirrer 1 and a collection bottle 2 placed above it, and the collection bottle 2 is connected to the exhaust pipe of the condenser 5 via a silicone tube. When installing the aeration plate 9, the fermentation tank cover 4 is opened, and all electrodes are removed before installation. The aeration plate 9 is placed inside the fermentation tank of the fermentation tank coupled foam separation device, and the aeration plate 9 vent hole and the feed port 21 are connected and fixed with a silicone tube. Feeding is no longer performed at the feed port 21, but is connected to an external gas device. During the separation process, the fermentation tank's own air inlet 15 is clamped with a clamp. The collection flask 2 is installed by attaching a double-hole lid to the exhaust pipe. One opening of the double-hole lid is connected to the exhaust pipe on the condenser 5 via a silicone tube, while the other opening is open. The collection flask 2 is placed on a magnetic stirrer 1, and a magnetic rotor is installed inside the flask. During the foam separation process, foam is generated in the tank and exits from the exhaust port 14. It is then passed through the silicone tube connected to the exhaust pipe of the condenser 5 into the collection flask 2, and then defoamed by the magnetic stirrer 1.

[0045] In the specific implementation process, the aeration plate 9 is composed of an aeration stone and a vent pipe. The diameter of the aeration stone is 5.8 cm, the outer diameter of the vent pipe is 6 mm, the pore size of the aeration stone is about 100 μm, and the number of pores on the aeration stone is calculated according to the area of the aeration stone. The bottom area is 8.41πcm 2 .

[0046] Example 2:

[0047] This example takes β-glucosidase expressed in Pichia pastoris as an example, and uses the internal fermentation tank coupled with foam separation device described in Example 1 for concentration and separation, thereby exploring the efficiency of foam separation of the fermentation broth under different conditions.

[0048] (1) Fermentation broth preparation:

[0049] Obtaining a recombinant expression strain: Using β-glucosidase as a model, a GB & his dual-tagged recombinant β-glucosidase (Glu-his-Linker-GB) was designed, and its nucleotide sequence is shown in SEQ ID NO: 1. Using the Pichia pastoris expression system, Glu-his-Linker-GB and the vector pPIC9K were double-digested with EcoRI and NotI, respectively. The double-digested fragments of Glu-his-Linker-GB and pPIC9K were then ligated with T4 DNA ligase to construct the recombinant plasmid pPIC9K-Glu-his-linker-GB. This recombinant plasmid was then transformed into competent Escherichia coli DH5α cells for replication and amplification, and then plated on LB plates containing the antibiotic ampicillin. A single colony was inoculated into 5 mL of LB medium containing 50 mg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm. The plasmid was then extracted, linearized with SacI, and transformed into competent Pichia pastoris KM71 (Accession No. cgmccno.14575). Culture was performed at 30°C for 3-5 days until a single colony emerged. Well-grown colonies were selected and plated onto YPD plates containing 0.25 mg / mL to 4.0 mg / mL G418 (Geneticin) in a sequential order from high to low concentrations. Cultured in a 30°C incubator for 2-3 days, and then single colonies were selected and stored in glycerol stock for future use.

[0050] SEQ ID NO: 1

[0051]

[0052] Seed solution preparation: Pick glycerol bacteria and streak them on a YPD plate. Incubate in a 30°C incubator for 2 days. Then pick a single colony and inoculate it into a conical flask containing 30 mL of YPD liquid medium. Incubate it in a shaking incubator at 30°C and 200 rpm for 24 hours. Then, inoculate it into a conical flask containing 100 mL of YPG medium at a ratio of 2.5% and incubate it in a shaking incubator at 30°C and 200 rpm for 24 hours.

[0053] High-density fermentation: 5% seed solution was inoculated into a fermenter containing 1.9 L of pH-adjusted BSM medium. The fermentation speed was set at 500 rpm, the temperature at 28°C, and the dissolved oxygen level at 30%. Automatic alkali replenishment (the reaction product tends to be acidic) was activated to adjust the pH to 6 in real time. BSM medium itself contains glycerol, and after inoculation, the first stage of fermentation, glycerol batch fermentation, began. The dissolved oxygen content at the beginning of bacterial inoculation is 100%. As the bacteria grow, the dissolved oxygen content gradually decreases. When the dissolved oxygen content drops to 20%-30%, the air intake and rotation speed are adjusted to keep the dissolved oxygen content within this range; then enter the glycerol feeding fermentation stage. After the bacteria continue to grow and consume glycerol, the dissolved oxygen content rises rapidly. At this time, glycerol is immediately supplemented. In this stage, the feeding rate is adjusted to keep the dissolved oxygen at around 20%-30%, and samples are taken at regular intervals to measure the OD600. After the OD600 reaches the preset value of 100, the addition of glycerol is stopped; finally, enter the carbon source feeding stage to induce fermentation. In this process, the dissolved oxygen will rise as the glycerol feeding is stopped. After 0.5-1h of starvation fermentation, methanol and 5% sorbitol (V Methanol :V Sorbitol =1:1), during which the dissolved oxygen was controlled at 20%-30%. After the expression level reached the maximum, the fermentation was stopped and subsequent experiments were carried out.

[0054] (2) The influence of different fermentation conditions on the foam separation effect during the internal coupled foam separation process of the fermenter:

[0055] This step investigates the effect of fermentation broth under different conditions on the foam separation effect inside the fermenter. The investigation steps are as follows: explore the effect of different initial protein concentrations, concentrations of added surfactants, and temperatures on the separation effect of the target protein in the fermentation broth, and collect the enrichment ratio E of the target protein in the foam. p , recovery rate R p and enzyme activity recovery rate R e The size is used to judge the separation effect, E p 、R p 、R e The calculation formula is as follows:

[0056]

[0057] Among them C f、V f and A f represent the protein concentration (mg / mL), volume (mL) and specific enzyme activity (U / mg) of the fermentation broth before foam separation; C i 、V i and A i represent the protein concentration (mg / mL), volume (mL) and specific enzyme activity (U / mg) of the defoaming solution after foam separation, respectively.

[0058] The specific process is as follows:

[0059] The fermentation tank is filled with 2L of fermentation broth with initial protein concentrations of 0.05mg / mL, 0.1mg / mL, and 0.15mg / mL, respectively, and the foam separation effect at a temperature of 25°C is investigated; after determining the optimal initial protein concentration, the separation and enrichment efficiency of the recombinant protein is explored when the concentrations of the added surfactant Tween 80 are 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, 1mg / mL, 1.2mg / mL, and 1.5mg / mL, respectively, while other conditions remain unchanged. After determining the optimal initial protein concentration and the concentration of the added surfactant, the fermentation broth temperature is adjusted to 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, while other conditions remain unchanged, to explore the effect of temperature on the separation and enrichment of the recombinant enzyme. The results are shown in Table 2. Figure 3 .

[0060] like Figure 3 As shown, Figure 3 A is the result of coupled foam separation inside the fermenter at different initial protein concentrations. As can be seen from the figure, with the increase of protein concentration, the enrichment ratio, recovery rate and enzyme activity recovery rate only changed slightly. The enrichment ratio showed a slight downward trend, while the enzyme activity recovery rate showed a slight upward trend. The recovery rate reached its maximum value when the protein concentration was 0.1 mg / mL. This may be because with the increase of protein concentration, the liquid holding capacity in the solution increased, resulting in a decrease in the enrichment ratio and an increase in the enzyme activity recovery rate. Finally, considering the changes in the enrichment ratio, recovery rate and enzyme activity recovery rate, the optimal initial protein concentration was determined to be 0.1 mg / mL. Figure 3 Figure B shows the effect of varying surfactant concentration on foam separation. Within the surfactant concentration range of 0.4-1.2 mg / mL, the recovery rate and enzyme activity recovery rate show a clear upward trend, while the enrichment ratio shows a clear downward trend. Adding surfactant during foam separation can improve the adsorption efficiency of proteins on the foam surface, but increasing surfactant concentration causes a large amount of liquid to enter the foam, resulting in a decrease in the enrichment ratio and an increase in the recovery rate. Combined with the changes in enrichment ratio, recovery rate, and enzyme activity recovery rate shown in the figure, the optimal surfactant addition level is determined to be 1.0 mg / mL. Figure 3Figure C shows the effect of temperature on foam separation. The figure shows an increasing trend in both recovery and enzyme activity recovery with increasing temperature, with significant increases within the 20-35°C temperature range. The enrichment ratio does not change significantly within the 20-35°C range, but decreases from 1.61 to 1.25 when the temperature increases from 35°C to 40°C. This is because increasing temperature facilitates the formation of bubbles in the solution, increasing the rate and number of bubbles produced per unit time and the foam holdup. Consequently, the recovery rate increases while the enrichment ratio decreases. Data comparison determined that 35°C was the optimal temperature for foam separation. The optimal initial protein concentration for foam separation in the fermenter was determined to be 0.1 mg / mL, the optimal surfactant addition level was 1.0 mg / mL, and the optimal temperature was 35°C. Under these optimal conditions, the enrichment ratio, recovery rate, and enzyme activity recovery after foam separation reached 1.61, 50.41%, and 61.04%, respectively.

[0061] Example 3:

[0062] This example examines the separation effects of different batches of fermentation broth under optimal foam separation conditions to determine the separation efficiency of fermentation-coupled foam separation. The specific implementation process of this example includes two points:

[0063] (1) Semi-continuous batch fermentation: The fermentation broth after 96 hours of fermentation was used as the seed liquid. At 96 hours of fermentation, part of the fermentation broth was taken out and stored for later use. Fresh BSM medium without glycerol was added to the remaining fermentation broth for fermentation. This process was repeated five times. The results were as follows: Figure 4 shown.

[0064] exist Figure 4 As the number of fermentation batches increased, the enzyme activity of the resulting product increased from an initial 1.18 U / mL to 3.52 U / mL after 96 hours of fermentation. After the third batch, the enzyme activity at 96 hours of fermentation reached 3.59 U / mL and 3.52 U / mL, respectively, indicating a stable state. Due to cell aging, the fermentation was repeated only five times.

[0065] (2) This step mainly involves foam separation of the first, second, and third batches of semi-continuous fermentation products through an in-tank foam separation coupling device. Since subsequent fermentation tends to be stable, the first three batches of samples are selected for analysis of the efficiency of the in-fermentation tank coupled foam separation. The foam separation conditions refer to those obtained in Example 2. Considering that the enzyme activity after separation is one of the main reference quantities, this section adds the fusion enzyme enrichment ratio E e The parameters are calculated as follows:

[0066]

[0067] Among them Af 、C f 、A i 、C i The representative content is consistent with that in Example 2.

[0068] The foam separation effect under different batches was investigated, and the results are shown in Table 1:

[0069] Table 1. Foam separation effects of different batches

[0070]

[0071] The results of foam separation are shown in Table 1 below. From Table 1, it can be seen that the second batch of fermentation has a higher enzyme activity of 1.21U / mL at the same protein concentration. After foam separation and defoaming, the enzyme activity is 1.76U / mL, which is nearly 1.5 times the initial enzyme activity. The enzyme activity in the remaining fermentation liquid after defoaming is only 0.37U / mL. At the same time, the E of foam separation is calculated. p 、E e 、R p 、R e The values of the second batch of fermentation were found to be 2.43, 3.52, 64.71% and 93.93% respectively. Compared with the results of the first batch of fermentation, the enrichment ratio and fusion enzyme enrichment ratio increased by 1.5 times, and the recovery rate and enzyme activity recovery rate increased by nearly 15-20%. At the same time, the effect of coupling foam separation of the products obtained from the third batch of fermentation was analyzed. From the results in the table, we can see that the E p 、E e 、R p 、R e The results were 2.39%, 3.19%, 70.94%, and 94.56%, respectively, similar to the results after foam separation of the second batch of products, demonstrating excellent separation results. Therefore, batch fermentation can also achieve separation of recombinant enzymes by coupling foam separation within the fermentor. Batch fermentation not only reduces resource and time costs during the fermentation process, but also exhibits excellent results in the subsequent concentration and separation of fermentation products coupled with foam separation.

[0072] In summary, the present invention uses a device coupled with foam separation inside a fermenter to directly perform foam separation on recombinant proteins inside the fermenter. The enrichment ratio of the separated protein can reach nearly 2.4, and the recovery rate can reach about 70%, with good separation effect. The method not only saves production costs, maintains the activity of the separated protein, and reduces protein loss during the separation process, but also has the advantages of simple separation equipment and the ability to separate and concentrate low-concentration proteins in large quantities. The method can solve the problems of possible denaturation of separated proteins, loss of enzyme activity, and high costs in the prior art, and has a significant effect on the separation and concentration of recombinant proteins.

[0073] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A device for coupling foam separation inside a fermentation tank, characterized in that: The device comprises: a fermentation tank body (3), and a fermentation tank cover (4) and a fermentation tank bottom plate (11) respectively connected to the fermentation tank body (3); The fermentation tank cover (4) is provided with a stirring shaft (7) with stirring blades (8) on one side in contact with the fermentation tank body (3), and a motor (6) for driving the stirring shaft (7) and the stirring blades (8) to rotate on the other side; the fermentation tank cover (4) is provided with a plurality of electrode interfaces; the fermentation tank cover (4) is also provided with a sample outlet (13), a sample inlet (20), a sampling port (12), an exhaust port (14), an air inlet (15) and a feed port (21); A stainless steel heating jacket (10) is provided below the fermentation tank body (3), and the stainless steel heating jacket (10) is connected to a water pipe (22); The fermentation tank chassis (11) comprises a chassis tray (24) supporting the bottom of the fermentation tank and a heating chassis (25) located above the chassis tray (24); the chassis tray (24) is provided with a port (27) connected to an external control device.

2. The device for coupling foam separation inside a fermentation tank according to claim 1, characterized in that: The fermentation tank body (3) is connected to the fermentation tank cover (4) through threads; the fermentation tank body (3) is connected to corresponding concave ports (26) on the chassis tray (24) through multiple convex ports (23) at the bottom of the fermentation tank body (3).

3. The device for coupling foam separation inside a fermentation tank according to claim 1, characterized in that: The electrode interfaces are arranged around the motor (6), and the electrode interfaces include a dissolved oxygen electrode interface (19), a pH electrode interface (16), a temperature electrode interface and (18) a defoaming electrode interface (17); the dissolved oxygen electrode interface (19), the pH electrode interface (16), the temperature electrode interface (18) and the defoaming electrode interface (17) are respectively connected to the dissolved oxygen electrode, the pH electrode, the temperature electrode and the defoaming electrode.

4. The device for coupling foam separation inside a fermentation tank according to claim 1, characterized in that: A condenser (5) connected to the exhaust port (14) is also installed above the fermentation tank cover (4); the condenser (5) includes a condenser water inlet pipe, a condenser water outlet pipe and an exhaust pipe.

5. The device for coupled foam separation inside a fermentation tank according to claim 1, characterized in that: During the foam separation process, an aeration plate (9) is provided inside the fermentation tank of the fermentation tank coupled with the foam separation device, and the aeration plate (9) is connected to the feed port (21) through a vent pipe (22); The fermentation tank internally coupled with the foam separation device is further provided with a magnetic stirrer (1) and a collecting bottle (2) placed above the magnetic stirrer, and the collecting bottle (2) is connected to the exhaust pipe of the condenser (5) through a silicone tube.

6. Use of the fermenter internal coupled foam separation device according to claim 1 in the production and separation of recombinant proteins.

7. The device for coupled foam separation inside a fermentation tank according to claim 6, characterized in that: The recombinant protein includes β-glucosidase.

8. A method for producing and isolating a recombinant protein, characterized in that: The method comprises: (1) Production of recombinant proteins: The seed liquid of the genetically engineered bacteria expressing the recombinant protein is inoculated into the device coupled with foam separation inside the fermentation tank according to claim 1, air is introduced into the device through the air inlet, the air intake is adjusted through the air inlet during the fermentation process, the carbon source is supplemented through the feed port, the state of the fermentation liquid during the fermentation stage is detected by a dissolved oxygen electrode, a pH electrode, and a temperature electrode, and the recombinant protein is produced by batch fermentation using a semi-continuous batch fermentation method to obtain a fermentation liquid; (2) Isolation of recombinant protein: A collecting bottle is connected to a foam separation device coupled inside the fermenter and the collecting bottle is placed on a magnetic stirrer. A feeding port is connected to an external gas device. An aeration disk is provided inside the fermenter of the device and the aeration disk is connected to the feeding port through a vent pipe. The fermentation liquid enters the device through an inlet, the inlet is closed, gas is blown into the device, foam is collected, and the magnetic stirrer defoams the collected foam, thereby achieving foam separation directly inside the fermenter to obtain separated and concentrated recombinant protein.

9. The method for producing and isolating a recombinant protein according to claim 8, characterized in that: In step (1), the fermentation liquid state during the fermentation stage is: dissolved oxygen 30%, temperature 28° C., and pH 6; The carbon source is glycerol before the OD600 value of the bacterial solution reaches 100, and methanol and sorbitol thereafter; During the semi-continuous batch fermentation, fermentation samples were collected and supplemented with BSM medium without glycerol; the BSM medium contained: 14.9 g of MgSO4·7H2O, 4.13 g of KOH, 0.93 g of CaSO4·2H2O, 18.2 g of K2SO4, 10 g of (NH4)2SO4, and 26.7 mL of H3PO4, and the volume was adjusted to 1 L with ddH2O.

10. The method for producing and isolating recombinant protein according to claim 8, characterized in that In step (2), during the foam separation process, the air blowing rate is controlled at the lowest range of the regulating valve of the external air inlet device, 1vvm; Adding surfactant Tween 80 to the fermentation broth; The protein concentration in the fermentation broth is 0.05-0.15 mg / mL; the final concentration of the surfactant Tween 80 is 0.4-1.5 mg / mL; The temperature of the fermentation liquid after gas injection is 20-40°C.

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