Catalytic enzyme protein purification method for ship carbon capture

Abstract

The invention provides a non-chromatography protein purification label and method suitable for a ship carbon capture biocatalyst-carbonic anhydrase. The method comprises the following steps: connecting a target protein with a purification tag through a proper connecting sequence-linker to construct a target protein-separation tag chimera, constructing the target protein-separation tag chimera in a proper escherichia coli expression vector, introducing the target protein-separation tag chimera into escherichia coli cells, carrying out conventional expansion and induction of strains, collecting bacterial liquid precipitate, and carrying out freeze-drying to obtain the target protein-separation tag chimera. The method comprises the following steps of: taking a crude enzyme as a raw material, resuspending the crude enzyme in a proper buffer solution, crushing at a certain power, collecting crude enzyme precipitate, resuspending the crude enzyme precipitate in the buffer solution, centrifuging at a low speed for 10 minutes, and discarding impurity supernatant, which is a round of purification process, and purifying for 2-3 rounds to obtain the target protein with higher purity.

Description

Technical Field

[0001] This invention belongs to the technical field of enzyme protein purification, specifically relating to a method for purifying catalytic enzyme proteins suitable for ship carbon capture. Background Technology

[0002] Carbon capture technology, as a key strategy for addressing global climate change and reducing greenhouse gas emissions, is undeniably important. With accelerated industrialization and continuous population growth, the concentration of carbon dioxide (CO2) in the atmosphere continues to rise, leading to a series of severe environmental problems such as global warming, frequent extreme weather events, glacial melting, and rising sea levels. Therefore, effectively controlling and reducing CO2 emissions is crucial for protecting the Earth's ecological environment and achieving sustainable development goals.

[0003] Carbonic anhydrase is a biological enzyme that can catalyze the reversible reaction between carbon dioxide (CO2) and water (H2O) with extremely high efficiency, producing bicarbonate ions (HCO3-). - CO2 and hydrogen ions (H⁺). This highly efficient catalytic ability enables the rapid absorption and conversion of CO2 under mild conditions during carbon capture, thereby improving capture efficiency.

[0004] Currently, there are many traditional methods for purifying proteases, both domestically and internationally, such as salting out, gel filtration, and ion exchange column chromatography. Although these techniques are mature, they often suffer from problems such as complex operation, long processing time, high cost, and limited purification efficiency. These drawbacks limit large-scale industrial applications, creating an urgent need for a purification method that is simple to operate, highly efficient, quick, and cost-effective. This paper designs a novel protease purification tag that enables efficient separation of carbonic anhydrase through simple purification steps, significantly shortening the purification cycle and reducing costs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a novel protease purification tag that is simple to operate, low in cost, and highly efficient in purification.

[0006] The purpose of this application is to provide a method for purifying catalytic enzyme proteins for ship carbon capture. It includes the following steps: (1) Construction of chimeric sequence: The target protein and the purification tag are linked by a linker to construct a target protein-separation tag chimera; (2) Plasmid synthesis: The chimera was constructed in a suitable E. coli expression vector; (3) Culture of bacterial strain: The expression vector was introduced into Escherichia coli cells and the bacterial strain was expanded and induced in a routine manner. The bacterial precipitate was collected and resuspended in a suitable buffer solution. (4) Protein extraction and purification: After crushing with a certain power, the crude enzyme precipitate is collected, then resuspended in buffer, centrifuged at low speed for 10 min, and the supernatant is discarded. This is one round of purification. After 2-3 rounds of purification, the target protein active precipitate with high purity can be obtained. (5) Target protein verification: The purity and size of the target protein are verified by electrophoresis; (6) Determination of the activity of the target protein.

[0007] In step (1), the target protein for constructing the chimera is PET degrading enzyme, lipase, methane monooxygenase, methanol dehydrogenase, pectin methyl esterase, and carbonic anhydrase, with carbonic anhydrase being preferred; the purification tag in step (1) is a protein with self-aggregating properties constructed from a ferritin tag sequence: MASISEKMVEALNRQINIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSERGGRVKLYAVEEPPSEWDSPLAAEHEVNVTKRIHELVEMAMQEKDFATYNFLQWYVAEQVSALDIVEKLRLIGEDAAALLFLDKELSLRQFTPPAEEEK.

[0008] Further, step (2) involves transferring the chimera from step (1) into the pET-22b(+) or pET-28a(+) expression vector; step (3) specifically involves: plasmid transformation: ① Take out the EP tube containing competent E. coli BL21(DE3) cells from the -80℃ ultra-low temperature freezer, quickly insert it into ice, and after the bacteria are almost thawed, inject the target DNA, gently stir the EP tube to mix it, and let it stand in ice for 25 minutes; ② Heat shock in a 42℃ water bath for 45 seconds, then quickly insert it into ice to stand. 3. Add 700 μL of antibiotic-free sterile LB medium to the EP tube, mix well, and revive for 1 h at 37°C and 200 rpm in a shaker; 4. Centrifuge the revived bacteria at 5000 rpm for 1 min, remove 900 μL of supernatant, mix the remaining liquid with the bacteria, and inoculate onto LB solid medium containing ampicillin resistance. Finally, invert the inoculated plate in a 37°C incubator for overnight culture; Step (3) also includes the preservation of the bacterial strain: pick a single colony from the overnight culture plate with an inoculation loop. The culture was transferred to LB medium containing 100 μg / mL ampicillin-resistant culture and cultured at 37°C and 200 rpm for 8-10 h. The cultured culture was then transferred to a new LB medium containing ampicillin and cultured again under the same conditions for 9-10 h. Finally, 1 mL of the cultured culture after two screenings was mixed with 330 μL of 80% glycerol culture, and the prepared glycerol culture was stored in an ultra-low temperature freezer at -80°C. Step (3) also includes the activation of the strain: the ultra-clean working... After cleaning the workbench, place 20 mL of LB medium in it and sterilize with UV light for 20 min. Add 20 μL of ampicillin resistance solution (100 g / L) to the LB medium, then add 200 μL of the above glycerol-bacterial mixture, mix well, and incubate overnight in a constant temperature shaker at 37°C and 200 rpm. Step (3) also includes expanding the culture and inducing protein expression: After cleaning the ultra-clean workbench, place 200 mL of TB medium in it and sterilize with UV light for 20 min. Add 20 mL of TB medium to the TB medium. Add 0.02-0.1 mol / L phosphate buffer, then add 200 μL of ampicillin resistance solution (100 g / L); finally add 2 mL of the overnight cultured bacterial solution, mix well, and incubate at 37°C and 200 rpm in a constant temperature shaker; when the OD600 value of the bacterial solution reaches 0.6-0.8, add 1 mL of 100 mmol / L LIPTG solution to the culture medium, mix well, and incubate at 18°C ​​and 180 rpm in a constant temperature shaker for 18-24 h.

[0009] Further, step (4) specifically involves: collection and disruption of bacterial cells: collecting the expressed bacterial cells from the culture medium in step (3) and aliquoting them into centrifuge bottles, centrifuging at 4000 rpm for 20 min at room temperature; discarding the supernatant in the centrifuge bottle after centrifugation, and adding pre-cooled pH 8.0, 50 mmol / L Tris-HCl buffer to the precipitate at a buffer-to-bacterial volume ratio of 1:20, gently mixing the bacterial precipitate in the buffer solution; and sonicating the resuspended bacterial solution under ice bath conditions. The ultrasonic power is 300-450W, the ultrasonic treatment is 2s, the pause is 4s, and the total working time is 20-35min; the step (4) also includes the low-speed centrifugation purification of recombinant protein: the solution containing cell fragments is centrifuged at 4℃ with a centrifugal force of 1000-5000×g for 10min; the precipitate obtained after two centrifugation and washing under the same conditions is the purified target protein; then we freeze-dry the purified target protein with a freeze dryer to obtain the target protein freeze-dried powder, and then store it in a refrigerator at -20℃.

[0010] Furthermore, step (5) specifically involves: mixing 10 μL of the above-mentioned target protein lyophilized powder sample buffer with 2.5 μL of protein loading buffer, then boiling it in 100°C water for 5 minutes. After boiling, the sample is centrifuged and mixed using a microcentrifuge. Electrophoresis buffer is added to the electrophoresis tank, and the comb on the gel plate is carefully removed. 5 μL of protein standard and 10 μL of the above-mentioned target protein lyophilized powder mixture are added to the loading wells of the gel plate, respectively. The power is turned on, and the voltage is set to 80V. Electrophoresis begins. When the protein band reaches the stacking gel, the voltage is increased to 120V. After electrophoresis, the gel is carefully separated from the glass plate and stained with rapid protein staining solution for 15-20 minutes. The stained gel is placed in deionized water and destained in a decolorizing shaker. After the water color deepens, the deionized water is replaced and the decolorization is repeated. Finally, the destained gel is observed and photographed under transmitted light using a GIS-2008 gel imaging system.

[0011] Further, the target protein activity determination in step (6) is as follows: 2 mL of ice-cold 10-35 mmol / L pH 8.3 Tris-HCl buffer, 20 μL of the mixture of 0.05-0.3 mg / ml of the target protein lyophilized powder obtained in step (4) after purification and dissolved in the buffer, and 0.2 mL of 0.05% bromothymol blue (BTB) indicator are added to a 4 mL glass cuvette and mixed thoroughly; the reaction is triggered by adding 1 mL of ice-cold saturated CO2 aqueous solution, and the reaction is carried out at 0°C; the time required for the solution to change from blue pH 8.3 to yellow pH 6.3 is recorded using a timer; enzyme activity is defined according to Wilbur Anderson's method, and one Wilbur Anderson unit is defined as: WAU = (Tc − Te) / Te, where Tc is the color change time of the solution without enzyme in seconds, and Te is the color change time of the solution containing enzyme in seconds; the hydration specific activity is the hydration activity value per mg of enzyme, in wau / mg.

[0012] The technical concept of this invention is as follows: This invention provides a non-chromatographic protein purification tag and method suitable for carbon capture biocatalysts in ships—carbonic anhydrase. The method involves linking the target protein and the purification tag using a suitable linker sequence to construct a target protein-separation tag chimera. This chimera is then constructed in a suitable *E. coli* expression vector, introduced into *E. coli* cells, and subjected to routine amplification and induction. The bacterial precipitate is collected and resuspended in a suitable buffer solution. After disruption at a certain power, the crude enzyme precipitate is collected, resuspended in the buffer solution, and centrifuged at low speed for 10 minutes. Impurities and supernatant are discarded. This constitutes one round of purification. After 2-3 rounds of purification, a target protein with high purity can be obtained. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the target protein and the purified tag chimera. Figure 2 This is a schematic diagram of the purification method; Figure 3 Electrophoresis was used to verify the purity of the target protein for carbonic anhydrase. The left image shows the electrophoresis of the undiluted purified protein, and the right image shows the electrophoresis of the diluted protein. Bands 1 and 2 are the crude enzyme bands before purification, and band 3 is the target band after purification. Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments: (1) Constructing a chimeric sequence: The target protein and the purification tag are linked by a linker to construct a target protein-separation tag chimera; the target protein for constructing the chimera is PET degrading enzyme, lipase, methane monooxygenase, methanol dehydrogenase, pectin methyl esterase, carbonic anhydrase, etc., preferably carbonic anhydrase.

[0015] (2) Plasmid synthesis: The chimera was constructed in a suitable E. coli expression vector; Detailed procedure: The chimera from step (1) is transferred into the pET-22b(+) or pET-28a(+) expression vector.

[0016] (3) Culture of bacterial strain: The expression vector in step (2) is introduced into Escherichia coli cells and the bacterial strain is expanded and induced in a conventional manner. The bacterial precipitate is collected and resuspended in a suitable buffer solution. Detailed Procedure: Plasmid Transformation: ① Remove the EP tube containing competent E. coli BL21 (DE3) cells from the -80℃ ultra-low temperature freezer and quickly place it on ice. After the bacteria are almost thawed, add the target DNA, gently stir the EP tube to mix, and incubate on ice for 25 min. ② Heat shock in a 42℃ water bath for 45 s, then quickly place it on ice for 2 min. ③ Add 700 μL of antibiotic-free sterile LB medium to the EP tube, mix well, and incubate at 37℃, 200 rpm for 1 h on a shaker. ④ Centrifuge the incubated cells at 5000 rpm for 1 min, remove 900 μL of supernatant, mix the remaining liquid with the cells, and inoculate onto LB solid medium containing ampicillin-resistant culture medium. Finally, invert the inoculated plate and incubate overnight at 37℃.

[0017] Preservation of the bacterial strain: Pick a single colony from an overnight plate using an inoculation loop and transfer it to LB medium containing 100 μg / mL ampicillin resistance. Incubate at 37°C and 200 rpm for 8-10 hours. Then transfer the cultured bacterial solution to a new LB medium containing ampicillin and incubate again under the same conditions for 9-10 hours. Finally, mix 1 mL of the bacterial solution that has undergone two screenings with 330 μL of 80% glycerol bacterial solution and store the prepared glycerol bacterial solution in an ultra-low temperature freezer at -80°C.

[0018] Activation of the bacterial strain: After cleaning the laminar flow hood, place 20 mL of LB medium in it and sterilize with UV light for 20 min. Add 20 μL of ampicillin resistance solution (100 g / L) to the LB medium, then add 200 μL of the above glycerol bacterial mixture, mix well, and incubate overnight in a constant temperature shaker at 37°C and 200 rpm.

[0019] Expanding the culture and inducing protein expression: After cleaning the laminar flow hood, place 200 mL of TB culture medium inside and sterilize under UV light for 20 min. Add 20 mL of 0.02-0.1 mol / L phosphate buffer to the TB culture medium, then add 200 μL of antibiotic solution (100 g / L), and finally add 2 mL of overnight cultured bacterial suspension. Mix well and incubate at 37°C and 200 rpm in a constant temperature shaker. When the OD600 value of the bacterial suspension reaches 0.6-0.8, add 1 mL of IPTG solution (100 mmol / L) to the culture medium, mix well, and incubate at 18°C ​​and 180 rpm in a constant temperature shaker for 18-24 h.

[0020] (4) Protein extraction and purification: After crushing with a certain power, the crude enzyme precipitate is collected, then resuspended in buffer, centrifuged at low speed for 10 min, and the supernatant is discarded. This is one round of purification. After 2-3 rounds of purification, the target protein active precipitate with high purity can be obtained. Detailed Procedure: Collection and Disruption of Bacterial Cells: Collect the expressed bacterial cells from the culture medium in step (3) and aliquot them into centrifuge bottles. Centrifuge at 4000 rpm for 20 min at room temperature. After centrifugation, discard the supernatant in the centrifuge bottle and add pre-cooled Tris-HCl buffer (pH 8.0, 50 mmol / L) to the precipitate at a buffer-to-bacterial volume ratio of 1:20. Gently pipette the bacterial precipitate in the buffer to mix it. Disrupt the resuspended bacterial solution by sonication in an ice bath at a power of 300-450 W for 2 s, followed by a 4 s pause. The total working time is 20-35 min.

[0021] Low-speed centrifugation purification of recombinant protein: The solution containing cell debris was centrifuged at 1000–5000 × g for 10 min at 4 °C. The precipitate obtained after two centrifugation washes under the same conditions was the purified target protein. The purified target protein was then lyophilized using a freeze dryer to obtain lyophilized protein powder, which was then stored at -20 °C.

[0022] Target protein verification: The purity and size of the target protein are verified by electrophoresis. Detailed instructions: Mix 10 μL of the above-mentioned lyophilized protein sample buffer with 2.5 μL of protein loading buffer, then boil in 100℃ water for 5 min. After boiling, centrifuge to mix the sample. Add 1× electrophoresis buffer to the electrophoresis tank, carefully remove the comb from the gel plate, and add 5 μL of protein marker and 10 μL of the above-mentioned lyophilized protein sample mixture to the loading wells of the gel plate. Turn on the power and set the voltage to 80V to start electrophoresis. When the protein bands reach the stacking gel, increase the voltage to 120V. After electrophoresis, carefully separate the gel from the glass plate and stain it with rapid protein staining solution for 15-20 min. Place the stained gel in deionized water on a destaining shaker for destaining. After the water color deepens, replace with fresh deionized water and repeat the destaining process. Finally, use a GIS-2008 gel imaging system to observe and photograph the destaining gel under transmitted light.

[0023] (6) Target activity assay: Taking the hydration activity test of carbonic anhydrase chimera as an example: 2 mL of ice-cold Tris-HCl buffer (10-35 mmol / L, pH 8.3), 20 μL of the 0.05-0.3 mg / ml target protein lyophilized powder obtained in step (4) above after purification dissolved in the buffer, and 0.2 mL of 0.05% bromothymol blue (BTB) indicator were added to a 4 mL glass cuvette and mixed thoroughly. The reaction was initiated by adding 1 mL of ice-cold saturated CO2 aqueous solution and carried out at 0 °C. The time required for the solution to change from blue (pH=8.3) to yellow (pH=6.3) was recorded using a timer. Enzyme activity was defined according to Wilbur Anderson's method. One Wilbur Anderson unit is defined as: WAU=(Tc−Te) / Te, where Tc is the color change time of the solution without enzyme (in seconds), and Te is the color change time of the solution containing enzyme (in seconds). Hydration specific activity is the hydration activity value per mg of enzyme, expressed in wau / mg.

[0024] The self-aggregating property of the purification tag enables the overexpression of the target protein, thereby achieving low-speed centrifugation purification. The target protein is in an active precipitate form. This non-chromatographic purification method has the advantages of being simple, efficient, low-cost, and easy to scale up.

[0025] Examples of using Escherichia coli to express carbonic anhydrase (1) Construction of chimeric sequence: The target protein and the purification tag are linked by a linker to construct a target protein-separation tag chimera; the target protein is carbonic anhydrase. The purification tag is a protein with self-aggregating properties constructed by the ferritin tag sequence: MASISEKMVEALNRQINIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSERGGRVKLYAVEEPPSEWDSPLAAEHEVNVTKRIHELVEMAMQEKDFATYNFLQWYVAEQVSALDIVEKLRLIGEDAAALLFLDKELSLRQFTPPAEEEK.

[0026] (2) Plasmid synthesis: The chimera was constructed in a suitable E. coli expression vector; Detailed procedure: The chimera from step (1) is transferred into the pET-22b(+) expression vector.

[0027] (3) Culture of bacterial strain: The expression vector in step (2) is introduced into Escherichia coli cells and the bacterial strain is expanded and induced in a conventional manner. The bacterial precipitate is collected and resuspended in a suitable buffer solution. Detailed Procedure: Plasmid Transformation: ① Remove the EP tube containing competent E. coli BL21 (DE3) cells from the -80℃ ultra-low temperature freezer, quickly insert it into ice, and after the bacteria are almost thawed, add the target DNA. Gently stir the EP tube to mix it, and incubate on ice for 25 min. ② Heat shock in a 42℃ water bath for 45 s, then quickly insert it into ice and incubate for 2 min. ③ Add 700 μL of antibiotic-free sterile LB medium to the EP tube, mix well, and incubate at 37℃, 200 rpm for 1 h on a shaker. ④ Centrifuge the incubated cells at 5000 rpm for 1 min, aspirate 900 μL of supernatant, mix the remaining liquid with the cells, and inoculate onto LB solid medium containing ampicillin resistance. Finally, invert the inoculated plate and incubate overnight at 37℃.

[0028] Preservation of the bacterial strain: Pick a single colony from an overnight plate using an inoculation loop and transfer it to LB medium containing 100 μg / mL ampicillin resistance. Incubate at 37°C and 200 rpm for 8-10 hours. Then transfer the cultured bacterial solution to a new LB medium containing ampicillin and incubate again under the same conditions for 9-10 hours. Finally, mix 1 mL of the bacterial solution that has undergone two screenings with 330 μL of 80% glycerol bacterial solution and store the prepared glycerol culture at -80°C.

[0029] Activation of the bacterial strain: After cleaning the laminar flow hood, place 20 mL of LB medium in it and sterilize with UV light for 20 min. Add 20 μL of ampicillin resistance solution (100 g / L) to the LB medium, then add 200 μL of the above glycerol bacterial mixture, mix well, and incubate overnight in a constant temperature shaker at 37°C and 200 rpm.

[0030] Expanding the culture and inducing protein expression: After cleaning the laminar flow hood, place 200 mL of TB medium inside and sterilize under UV light for 20 min. Add 20 mL of 0.02 mol / L sodium phosphate buffer to the TB medium, then add 200 μL of antibiotic solution (100 g / L), and finally add 2 mL of overnight cultured bacterial suspension. Mix well and incubate at 37°C and 200 rpm in a constant temperature shaker. When the OD600 value of the bacterial suspension reaches 0.6-0.8, add 1 mL of IPTG solution (100 mmol / L) to the medium, mix well, and incubate at 18°C ​​and 180 rpm for 24 h.

[0031] (4) Protein extraction and purification: After crushing with a certain power, the crude enzyme precipitate is collected, then resuspended in buffer, centrifuged at low speed for 10 min, and the supernatant is discarded. This is one round of purification. After three rounds of purification, the target protein active precipitate with high purity can be obtained. Detailed operation: Collection and disruption of bacterial cells: Collect the expressed bacterial cells from the culture medium in step (3) and aliquot them into centrifuge bottles. Centrifuge at 4000 rpm for 20 min at room temperature. After centrifugation, discard the supernatant in the centrifuge bottle and add pre-cooled Tris-HCl buffer (pH 8.0, 50 mmol / L) to the precipitate at a buffer-to-bacterial volume ratio of 1:20. Gently pipette the bacterial precipitate in the buffer to mix it. Sonicate the resuspended bacterial solution under ice bath conditions. The ultrasonic power is 300-450 W. Sonicate for 2 s, pause for 4 s, and the total working time is 30 min. Low-speed centrifugation purification of recombinant protein: Centrifuge the solution containing cell debris at 1000 × g for 10 min at 4 °C. The precipitate obtained after two centrifugations and washings under the same conditions is the purified target protein. Then, freeze-dry the purified target protein to obtain the target protein lyophilized powder and store it in a freezer at -20 °C.

[0032] Target protein verification: The purity and size of the target protein are verified by electrophoresis.

[0033] Detailed instructions: Mix 10 μL of the above-mentioned lyophilized protein sample buffer with 2.5 μL of protein loading buffer, then boil in 100℃ water for 5 min. After boiling, centrifuge to mix the sample. Add 1× electrophoresis buffer to the electrophoresis tank, carefully remove the comb from the gel plate, and add 5 μL of protein marker and 10 μL of the above-mentioned lyophilized protein sample mixture to the loading wells of the gel plate. Turn on the power and set the voltage to 80V to start electrophoresis. When the protein bands reach the stacking gel, increase the voltage to 120V. After electrophoresis, carefully separate the gel from the glass plate and stain with rapid protein staining solution for 20 min. Place the stained gel in deionized water on a destaining shaker for destaining. After the water color deepens, replace with fresh deionized water and repeat the destaining process. Finally, use a GIS-2008 gel imaging system to observe and photograph the destaining gel under transmitted light.

[0034] (6) Activity assay: Taking the hydration activity test of carbonic anhydrase chimera as an example: 2 mL of ice-cold Tris-HCl buffer (10 mmol / L, pH 8.3), 20 μL of the 0.1 mg / mL lyophilized target protein obtained in step (4) above after purification dissolved in the buffer, and 0.2 mL of 0.05% bromothymol blue (BTB) indicator were added to a 4 mL glass cuvette and mixed thoroughly. The reaction was initiated by adding 1 mL of ice-cold saturated CO2 aqueous solution and carried out at 0 °C.

[0035] Electrophoresis diagram Figure 3 The verification was performed by adding carbonic anhydrase to purify the tag chimera, which had a size of 49 kDa and a purity of over 85%.

[0036] Hydration activity test results showed that the hydration specific activity of the carbonic anhydrase chimera with this purification tag was approximately 900 wau / mg, which is about 1 / 10 of the activity of commercial carbonic anhydrase.

[0037] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for purifying catalytic enzyme proteins for ship carbon capture, characterized in that, Includes the following steps: (1) Construction of chimeric sequence: The target protein and the purification tag are linked by a linker to construct a target protein-separation tag chimera; (2) Plasmid synthesis: The chimera was constructed in a suitable E. coli expression vector; (3) Culture of bacterial strain: The expression vector was introduced into Escherichia coli cells and the bacterial strain was expanded and induced in a routine manner. The bacterial precipitate was collected and resuspended in a suitable buffer solution. (4) Protein extraction and purification: After crushing with a certain power, the crude enzyme precipitate is collected, then resuspended in buffer, centrifuged at low speed for 10 min, and the supernatant is discarded. This is one round of purification. After 2-3 rounds of purification, the target protein active precipitate with high purity can be obtained. (5) Target protein verification: The purity and size of the target protein are verified by electrophoresis; (6) Determination of the activity of the target protein.

2. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, The target proteins for constructing the chimera in step (1) are PET degrading enzyme, lipase, methane monooxygenase, methanol dehydrogenase, pectin methyl esterase, and carbonic anhydrase, with carbonic anhydrase being preferred.

3. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, The purification tag in step (1) is a protein with self-aggregating properties constructed by the ferritin tag sequence: MASISEKMVEALNRQINIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSERGGRVKLYAVEEPPSEWDSPLAAEHEVNVTKRIHELVEMAMQEKDFATYNFLQWYVAEQVSALDIVEKLRLIGEDAAALLFLDKELSLRQFTPPAEEEK.

4. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, Step (2) involves transferring the chimera from step (1) into the pET-22b(+) or pET-28a(+) expression vector.

5. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, The specific steps (3) are as follows: Plasmid transformation: ① Take out the EP tube containing competent E. coli BL21 (DE3) cells from the -80℃ ultra-low temperature freezer, quickly insert it into ice, and after the bacteria are almost thawed, add the target DNA. Gently tap the EP tube to mix it, and let it stand in ice for 25 min; ② Heat shock in a 42℃ water bath for 45 s, and then quickly insert it into ice and let it stand for 2 min; ③ Add 700 μL of antibiotic-free sterile LB medium to the EP tube, mix it, and then revive it in a shaker at 37℃ and 200 rpm for 1 h; ④ Centrifuge the reviving bacteria at 5000 rpm for 1 min, remove 900 μL of supernatant, mix the remaining liquid with the bacteria, and inoculate it onto LB solid medium containing ampicillin resistance. Finally, invert the inoculated plate in a 37℃ incubator for overnight culture.

6. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 4, characterized in that, Step (3) also includes the preservation of the bacterial strain: pick a single colony from the overnight culture plate with an inoculation loop, transfer it to LB medium containing 100 μg / mL ampicillin-resistant culture medium, and culture it in a constant temperature shaker at 37°C and 200 rpm for 8-10 h; then transfer the cultured bacterial solution to a new LB medium containing ampicillin and culture it again under the same conditions for 9-10 h; finally, take 1 mL of the bacterial solution that has been screened twice and mix it with 330 μL of 80% glycerol bacterial solution, and store the prepared glycerol bacterial solution in an ultra-low temperature freezer at -80°C.

7. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 5, characterized in that, Step (3) also includes the activation of the strain: after cleaning the ultra-clean workbench, put in 20 mL of LB medium and turn on the ultraviolet sterilization for 20 min; add 20 μL of ampicillin resistance solution 100 g / L to the LB medium, then add 200 μL of the above glycerol bacterial mixture, mix well, and place in a constant temperature shaker at 37℃ and 200 rpm for overnight culture.

8. A method for purifying catalytic enzyme proteins for ship carbon capture according to claim 6, characterized in that, Step (3) further includes expanding the culture and inducing protein expression: After cleaning the ultra-clean workbench, place 200 mL of TB culture medium in it and turn on the UV sterilization for 20 min; add 20 mL of 0.02-0.1 mol / L phosphate buffer to the TB culture medium, and then add 200 μL of 100 g / L ampicillin resistance solution; finally, add 2 mL of the above overnight cultured bacterial solution, mix well, and place it in a constant temperature shaker at 37℃ and 200 rpm for culture; when the OD600 value of the bacterial solution reaches 0.6-0.8, add 1 mL of 100 mmol / L LIPTG solution to the culture medium, mix well, and place the culture medium in a constant temperature shaker at 18℃ and 180 rpm for 18-24 h.

9. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, The specific steps (4) are as follows: collection of bacterial cells and cell disruption: collect the expressed bacterial cells in the culture medium of step (3) and dispense them into centrifuge bottles. Centrifuge at 4000 rpm for 20 min at room temperature. After centrifugation, discard the supernatant in the centrifuge bottle and add pre-cooled pH 8.0, 50 mmol / L Tris-HCl buffer to the precipitate at a ratio of 1:20 between the buffer and the bacterial solution. Gently pipette the bacterial precipitate in the buffer solution to mix it. Sonicate the resuspended bacterial solution under ice bath conditions. The ultrasonic power is 300-450 W. Sonicate for 2 s, pause for 4 s, and the total working time is 20-35 min.

10. A method for purifying catalytic enzyme proteins for ship carbon capture according to claim 8, characterized in that, Step (4) also includes low-speed centrifugation purification of recombinant protein: the solution containing cell debris is centrifuged at 1000-5000×g for 10 min at 4°C; the precipitate obtained after two centrifugation and washing under the same conditions is the purified target protein. We then freeze-dried the purified target protein using a freeze dryer to obtain the target protein freeze-dried powder, and then stored it in a freezer at -20°C.

11. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, Step (5) is as follows: Mix 10 μL of the above-mentioned target protein lyophilized powder sample buffer with 2.5 μL of protein loading buffer evenly, then boil in boiling water at 100℃ for 5 min. After boiling, centrifuge the sample evenly using a microcentrifuge. Add electrophoresis buffer to the electrophoresis tank, carefully remove the comb from the gel plate, and add 5 μL of protein standard and 10 μL of the above-mentioned target protein lyophilized powder mixture to the loading well of the gel plate respectively. Turn on the power and set the voltage to 80V to start electrophoresis. When the protein band reaches the stacking gel, increase the voltage to 120V. After electrophoresis, carefully separate the gel from the glass plate and stain it with protein rapid staining solution for 15-20 min. Place the stained gel in deionized water and decolorize it in a decolorizing shaker. After the water color deepens, replace it with new deionized water and repeat the decolorization. Finally, use the GIS-2008 gel imaging system to observe and photograph the decolorized gel under transmitted light.

12. The method for purifying catalytic enzyme proteins for ship carbon capture according to claim 1, characterized in that, The specific steps (6) for determining the activity of the target protein are as follows: 2 mL of ice-cold 10-35 mmol / L pH 8.3 Tris-HCl buffer, 20 μL of a mixture of 0.05-0.3 mg / mL of the target protein lyophilized powder obtained in step (4) after purification, dissolved in the buffer, and 0.2 mL of 0.05% bromothymol blue (BTB) indicator are added to a 4 mL glass cuvette and mixed thoroughly. The reaction is initiated by adding 1 mL of ice-cold saturated CO2 aqueous solution, and the reaction is carried out at 0°C. The time required for the solution to change from blue (pH 8.3) to yellow (pH 6.3) is recorded using a timer. Enzyme activity is defined according to Wilbur Anderson's method. One Wilbur Anderson unit is defined as: WAU = (Tc − Te) / Te, where Tc is the color change time of the solution without enzyme, in seconds, and Te is the color change time of the solution containing enzyme, in seconds. The hydration specific activity is the hydration activity value per mg of enzyme, in wau / mg.

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