CN111518851B - Immobilized enzyme continuous preparation 14/15 N]Process for preparing L-citrulline - Google Patents

Abstract

The application discloses a continuous preparation of immobilized enzyme ^{14/ 15} N]The method of L-citrulline belongs to the field of enzymology and enzyme engineering technology. The method is that the fusion protein containing the immobilized enzyme is suspended in a packed bed reactor; will then contain [ [ ^14/15 N]The solution of the L-arginine flows through a packed bed reactor for reaction at the temperature of 20-55 ℃ and the flow rate of 0.3-0.5BV/h, and the reaction solution is separated and purified to obtain the [14/15N ]]-L-citrulline. The technical concept of the invention is to fix arginine deiminase arc on inclusion body protein cipA by adopting cipA immobilized fusion protein as a carrier to generate inclusion body protein cipA-arc with catalytic activity, namely cipA-arc fusion protein. Immobilized c with catalytic activity provided hereinipA-arginine deiminase cipA-arc fusion protein can continuously react for more than 560 hours; simultaneously obtained isotopically labelled [ ^14/15 N]L-citrulline provides an effective way for diagnosing and treating prostate diseases, cardiovascular diseases and the like.

Description

A method for continuously preparing [14/15N]-L-citrulline using immobilized enzyme

Technical Field

The present application relates to a method for producing high-purity [ ^14/15 N]-L-citrulline, in particular to a method for producing high-purity [ ^14/15 N]-L-citrulline by decomposing [ ^14/15 N]-L-arginine using recombinant arginine deiminase, belonging to the technical field of enzymology and enzyme engineering.

Background Art

L-citrulline is a special amino acid. It participates in many metabolic processes in the body, such as scavenging free radicals, being an indicator of allograft rejection, vasodilation, stabilizing blood pressure, diagnosing rheumatoid arthritis, anti-oxidation, preventing prostate disease, improving sexual function, anti-aging, and enhancing immunity, etc. It has a very broad application prospect.

There are three methods for producing L-citrulline: chemical method, fermentation method and enzyme method. The chemical method refers to the hydrolysis of L-arginine under alkaline conditions to obtain L-citrulline. The hydrolysis process is difficult to control, and the product contains the optical antipode D-citrulline, which affects the product quality. In addition, a large amount of wastewater is generated during the production process, polluting the environment. The difficulty of the fermentation method is that the yield of L-citrulline per unit volume is low, and the operation process of extracting L-citrulline from the fermentation broth is complicated, the yield is low, and the cost is high. The enzymatic production method refers to the conversion of L-arginine into L-citrulline under the action of arginine deiminase. This method has the advantages of strong specificity and high product concentration, but this method also has the problem of low catalyst utilization, that is, the bacterial cell must be fermented again after each production, which not only consumes a large amount of raw materials, but also generates a large amount of wastewater, increasing the cost of environmental protection treatment; and impurities (a large amount of bacterial cells, proteins and various metal ions remaining in the fermentation broth) brought into the reaction system by the enzyme catalyst result in a series of bacterial cell removal, protein removal and ion column removal process steps in the post-treatment process of L-citrulline, further increasing the production cost.

Since the production of L-citrulline by chemical, fermentation or enzymatic methods has many disadvantages, the production cost remains high, which brings great difficulties to its actual promotion and application.

In order to overcome these problems, solutions for immobilized enzymes or cells have been proposed. There are two major methods for preparing immobilized enzymes or cells: physical method and chemical method. Physical methods include physical adsorption method, embedding method, etc. The advantage of physical method for immobilizing enzymes is that the enzyme does not participate in chemical reactions, the overall structure remains unchanged, and the catalytic activity of the enzyme is well preserved. However, since the embedding material or semipermeable membrane has a certain spatial or stereoscopic hindering effect, it is not applicable to some reactions. Chemical methods include binding method and cross-linking method. In chemical methods, the enzyme is tightly bound to the carrier, not easy to fall off, and has good stability, but the reaction conditions are fierce, the operation is complicated, the control conditions are harsh, and the activity loss is large.

In 2008, Zheng Pu et al. (Zheng Pu, Ni Ye, Zhang Wen. Continuous preparation of L-citrulline by immobilized Pseudomonas cells in a packed bed reactor [J]. Journal of Food and Biotechnology, 2008, 27(5): 1673-1689) reported that the continuous preparation of L-citrulline by immobilized Pseudomonas cells in a packed bed reactor could be operated continuously for 54 days under the condition of 0.0108 g·(hg) ^-1 (grams of citrulline produced per gram of cells per hour). However, the bacterial fermentation production process is complicated, and the immobilization time of cells is too long, the substrate concentration is low, and the yield is not high. Although the immobilized cells are simple, there is still the problem of cell fragmentation and release of foreign proteins and other organic substances in the bacteria, which increases the difficulty of separation and purification of the product in the reaction system. In addition, cell immobilization also increases the number of operation steps and increases the production cost.

Summary of the invention

According to the first aspect of the present application, a method for continuously preparing [ ^14/15 N]-L-citrulline using an immobilized enzyme is provided, the method comprising the following steps:

(1) suspending a fusion protein containing an immobilized enzyme in a packed bed reactor;

(2) A solution containing [ ^14/ 15N]-L-arginine is passed through a packed bed reactor at a flow rate of 0.3-0.5 BV/h at 20-55°C.

Optionally, the fusion protein containing the immobilized enzyme in step (1) is 9000-12000U, preferably 10000U, and the concentration of [ ^14/ 15N]-L-arginine in the solution containing [ ^14/ 15N]-L-arginine in step (2) is 1.0-2.5mol/L.

Optionally, the packed bed reactor in step (1) is a glass column with a diameter-to-height ratio of 15-40 and a volume of 450-2000 mL.

Optionally, the concentration of [ ^14/15 N]-L-arginine in the solution containing [ ^14/15 N]-L-arginine is 1.0-2.5 mol/L.

Optionally, the fusion protein containing the immobilized enzyme in step (1) is the inclusion body protein cipA-arc with catalytic activity obtained by fixing arginine deiminase arc on the inclusion body protein cipA using cipA as a carrier, that is, the cipA-arc fusion protein.

Optionally, the cipA-arc fusion protein is prepared by the following steps:

(1) preparing Corynebacterium glutamicum competent cells;

(2) using the recombinant plasmid pXMJ19-cipA-arc to electroporate the competent cells of Corynebacterium glutamicum described in step (1) to obtain recombinant bacteria;

(3) The whole cells of the recombinant bacteria obtained in step (2) are induced to express by genetically engineered bacteria, and the resulting precipitate is the cipA-arc fusion protein after ultrasonic disruption and centrifugation.

Optionally, the Corynebacterium glutamicum competent cells are prepared by the following method:

After culturing Corynebacterium glutamicum ATCC13032 in a solid culture medium containing LBG, fresh strains are selected and inoculated into a LBG liquid culture medium, and after culturing, the activated bacterial solution is transferred to the LBG culture medium at an inoculation amount of 0.8-1.5%, and the culture is continued until the OD600 is 0.8-1.0; the bacterial solution is precooled and centrifuged with an ice-water mixture, and glycerol is added after the supernatant is sucked out, and the solution is pipetted until the bacteria are suspended, and the solution is centrifuged again, and glycerol is added after the supernatant is sucked out, and the solution is pipetted until the bacteria are suspended, so as to obtain Corynebacterium glutamicum competent cells.

As a preferred solution, Corynebacterium glutamicum ATCC13032 is streaked on a plate containing LBG solid medium, cultured in an incubator, and fresh strains are picked out after the bacteria grow out and inoculated into LBG liquid medium, and cultured in a shaking incubator at a temperature of 20-40°C and a rotation speed of 150-300 r/min for 12-24 hours; the activated bacterial liquid is transferred to the LBG medium at a 1% inoculation amount, and cultured in a shaking incubator at a temperature of 20-40°C and a rotation speed of 150-300 r/min until the OD600 is about 0. 9. Precool the bacterial solution in an ice-water mixture for 15-20 minutes, then dispense the precooled bacterial solution into sterilized centrifuge tubes in a clean bench, centrifuge at 4°C 6000g for 30 seconds, and place in ice water for 2 minutes; aspirate the supernatant in the centrifuge tube, add precooled 10% glycerol to each centrifuge tube, and slowly pipette until the bacteria are suspended; centrifuge the suspension at 4°C 6000g for 30 seconds, aspirate the supernatant in the centrifuge tube, add precooled 10% glycerol to each centrifuge tube, and slowly pipette until the bacteria are suspended.

Optionally, the culture temperature is 30° C. and the rotation speed during the culture is 200 r/min.

Optionally, the recombinant plasmid pXMJ19-cipA-arc electroporation transformation competent cells are prepared by the following method:

Take competent cells of Corynebacterium glutamicum and recombinant plasmid pXMJ19-cipA-arc and mix them evenly. After cooling on ice, perform electric shock at the same temperature with a voltage of 1-5 kV and an electric shock of 1-10 ms. Then add LBG liquid culture medium at room temperature, transfer to a centrifuge tube, and after shaking culture, take the obtained liquid and spread it on a chloramphenicol resistance plate, select a single colony to extract the plasmid, and then confirm the insertion of the target fragment through double enzyme digestion and PCR to obtain the recombinant bacteria inoculation.

As a preferred solution, the competent cells and the recombinant plasmid pXMJ19-cipA-arc are mixed and cooled on ice for 10 minutes; the cells are quickly placed in an ice-cold electric shock cup for electric shock at a voltage of 2-4 kV and a time of 3-7 ms; the electric shock cup is removed as soon as possible after the pulse ends, LBG liquid culture medium is added at room temperature, the cells are transferred to a centrifuge tube and gently shaken for culture for 2 hours, and the resulting liquid is spread on a 20 μg/mL chloramphenicol resistance plate; a single colony is selected to extract the plasmid, and the insertion of the target fragment is confirmed by double enzyme digestion or PCR.

Optionally, the voltage of the electric shock is 2.5 kV; the duration of the electric shock is 5 ms.

Optionally, it is characterized in that the induction expression method of the genetically engineered bacteria is as follows:

The recombinant bacteria were inoculated into LBG medium containing chloramphenicol, and isopropyl-β-D-thiogalactoside was added when the bacterial OD600 value reached 0.8-1.0 after shaking culture. After induction overnight, the whole cells of the recombinant bacteria were collected by centrifugation, the bacteria were washed with Tris-HCl buffer and resuspended in phosphate buffer, the cells were ultrasonically disrupted and centrifuged again, and the precipitate was the obtained cipA-arc fusion protein.

As a preferred solution, the successfully identified recombinant bacteria are inoculated into LBG medium containing chloramphenicol at a final concentration of 20 μg/mL, the culture temperature is set to 20-40°C, the shaker speed is 150-300 r/min, and IPTG is added at a final concentration of 1 mM when the OD600 value of the bacteria reaches 0.9, and the cells are induced overnight at a temperature of 20-40°C and a speed of 150-300 r/min; the recombinant bacterial cells are collected by centrifugation at 4°C, the bacteria are washed with buffer and resuspended in another buffer, the cells are ultrasonically broken and centrifuged again at 4°C, and the precipitate is the obtained cipA-arc fusion protein.

Optionally, the temperature of the culture and the induction are both 30°C; the rotation speed during the culture is 200r/min; and the rotation speed during the induction is 180r/min.

Optionally, the washing buffer is Tris-HCl; the resuspending buffer is a phosphate buffer;

Preferably, the pH value of Tris-HCl is 7.0;

Preferably, the pH value of the phosphate buffer is 6.5.

Optionally, arginine deiminase is expressed in the genetically engineered bacteria.

Optionally, the genetically engineered bacteria are constructed by the following method:

1) The cipA gene sequence was introduced into the HindIII site at the 5' end of the DNA and the SalI site at the 3' end to obtain a fragment with a gene sequence of SEQ ID NO.1. After sequencing, the synthesized fragment was double-digested with HindIII/SalI to form the target gene and the expression vector pXMJ19. After the digestion product was recovered by gel, the target fragment and the vector were connected. The connection product was transformed into Escherichia coli DH5α competent cells to obtain the positive transformant pXMJ19-cipA;

2) The arc gene sequence was introduced into the XhoI site at the 5' end of the DNA and the SacI site at the 3' end to obtain a fragment with a gene sequence of SEQ ID NO.2. After sequencing, the synthesized fragment was double-digested with XhoI/SacI to form the target gene and the expression vector pXMJ19-cipA. After the digestion product was recovered by gel, the target fragment and the vector were connected. The connection product was transformed into Escherichia coli DH5α competent cells to obtain the positive transformant pXMJ19-cipA-arc, which is a genetically engineered bacterium containing arginine deiminase.

The genetically engineered bacteria containing arginine deiminase is deposited under the name of Corynebacterium glutamicum SUMHS-2020.01 and is classified as Corynebacterium glutamicum. The strain was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China on January 17, 2020. The deposit number of the culture collection center is CGMCC No.19404.

Another technical solution of the present invention is to express arginine deiminase in the genetically engineered bacteria containing arginine deiminase.

Optionally, the solution containing [ ^14/ 15N]-L-arginine further contains ammonium acetate buffer solution, ammonium formate buffer solution, ammonium chloride aqueous solution, ammonium bicarbonate aqueous solution or aqueous solution.

Preferably, the solution containing [ ^14/ 15N]-L-arginine further contains ammonium acetate buffer solution, ammonium formate buffer solution, ammonium chloride aqueous solution or ammonium bicarbonate aqueous solution.

Optionally, the concentration of the ammonium acetate buffer solution is 0.2 mol/L, and the pH is 6.0; the concentration of the ammonium formate buffer solution is 0.2 mol/L, and the pH is 6.0; the concentration of the ammonium chloride aqueous solution is 0.3 mol/L, and the pH is 4.5; the concentration of the ammonium bicarbonate aqueous solution is 0.3 mol/L, and the pH is 8.5; and the pH of the aqueous solution is 7.5.

Optionally, the method further comprises separation and purification of [ ^14/ 15N]-L-citrulline.

Optionally, the separation and purification steps are as follows:

1) collecting the reaction liquid flowing out of the packed bed reactor, removing the buffer salt by nanofiltration, returning the reaction liquid to the reaction system, recycling it, and collecting the product concentrate;

2) The reaction solution after step 1) is concentrated under vacuum, crystallized and dried to obtain a white powdery solid, which is [ ^14/15 N]-L-citrulline with a purity of more than 99.5%.

In this application, "cipA gene sequence" refers to the cipA gene sequence reported by Kirsten Jung et al. (Wang Y, Heermann R, Jung K. CipA and CipB as Scaffolds To Organize Proteins into Crystalline Inclusions [J]. ACS Synthetic Biology, 2017, 6, 826-836).

In the present application, "arc gene sequence" refers to the arginine deiminase (arc) gene sequence reported by Kim et al. (Kim J E, Jeong D W, Lee HJ. Expression, purification, and characterization of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 7962 in Escherichia coli BL21 [J]. Protein Expression and Purification, 2007, 53 (1): 0-15).

In the present application, "pXMJ19" refers to a vector carrying a gene expression protein in Corynebacterium glutamicum.

The beneficial effects of this application include:

1) In this application, the gene of arginine deiminase was synthesized and cloned, an engineered bacterium with high arginine deiminase production was constructed, and arginine deiminase was expressed in Corynebacterium glutamicum;

2) The present application immobilizes arginine deiminase on the inclusion body protein cipA to produce the inclusion body protein cipA-arc (i.e., cipA-arc fusion protein) with catalytic activity. The immobilization method is simple, fast, low-cost, high-efficiency, and easy to use;

3) The immobilized cipA-arginine deiminase (cipA-arc) fusion protein with catalytic activity provided in the present application can react continuously for more than 560 hours;

4) The inclusion body protein cipA-arc provided in the present application, i.e., the arc-cipA immobilized fusion protein, can catalyze the conversion of [ ^14/15 N]-L-arginine into [ ^14/15 N]-L-citrulline. Through the fixed bed reactor, the post-treatment is simple, the product separation and purification is convenient, the cost is low, and it is easy to scale up the production, which adds a new way for the enzymatic preparation of [ ^14/15 N]-L-citrulline;

5) The isotope-labeled [ ^14/ 15N]L-citrulline provided in this application provides an effective approach for the diagnosis and treatment of prostate diseases, cardiovascular diseases, etc.

DETAILED DESCRIPTION

The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

Unless otherwise specified, the raw materials in the examples of this application were purchased from the exploration platform, among which the plasmid pXMJ19 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

Corynebacterium glutamicum ATCC13032 was purchased from Guangdong Microorganism Collection Center.

[ ^14/15 N]-L-citrulline determination method: The product [ ^14/15 N]-L-citrulline was determined by HPLC, the chromatographic column was C18, 5μm, 250mm×4.6mm; the mobile phase was 5% methanol; the flow rate was 1mL/min; the detection wavelength was 290nm; and the column temperature was room temperature.

Definition of cipA-arginine deiminase activity: Under the conditions of 37°C and pH 6.0, the amount of enzyme that catalyzes the conversion of [ ^14/15 N]-L-arginine to produce 1 μmol of [ ^14/15 N]-L-citrulline per minute is defined as one unit of enzyme activity (1U).

Definition of specific enzyme activity: the amount of enzyme activity contained in each mg of protein (U/mg). Protein concentration was determined using the Bradford method.

According to one embodiment of the present application, it mainly includes: 1) chemically synthesizing target genes (cipA, arc); 2) continuously connecting the synthesized cipA and arc to the vector pXMG19 to construct the expression vector pXMJ19-cipA-arc; 3) introducing pXMJ19-cipA-arc into Corynebacterium glutamicum ATCC13032 by electroporation; 4) inducing expression and isolating the inclusion body protein cipA-arc (i.e., cipA-arc fusion protein); 5) using the inclusion body protein cipA-arc to continuously catalyze arginine to synthesize [ ^14/15 N]-L-citrulline in a packed bed reactor.

In the examples of the present application, the [ ^14/15 N]L-citrulline conversion rate is calculated based on the carbon molar number.

Example 1 Construction of genetically engineered bacteria containing arginine deiminase

1.1 Based on the cipA gene sequence reported by Kirsten Jung et al. (2017), the coding region DNA optimized according to the codon preference of Corynebacterium glutamicum was chemically synthesized. The chemical synthesis was completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The cipA gene sequence is as follows:

ATGATCAACGACATGCACCCATCCCTGATCAAGGACAAGGACATGATGGACGACGTTATGCTGCGCTCCTGCAAGATCATCGCTATGAAGATCATGCCAGACAAGGTTATGCAGGTTATGGTTACCGTTCTGATGCTGGACGGCACCTCCGAGGAGATGCTGCTGAAGTGGAACCTGCTGGACAACCGCGGCATGGCTATCTACAAGGTTCTGATGGAGGCTCTGTGCGGCAAGAAGGACGTTTAAGATCGGCACCGTT GGCAAGGTTGGCCCACTGGGCTGCGACTACATCAACTGCGTTGAGATCTCCATG

The synthesized gene sequence (SEQ ID NO.1) introduced a HindIII site at the 5' end of the DNA and a SalI site at the 3' end. After sequencing, the synthesized fragment was double-digested with HindIII/SalI to form the target gene and expression vector pXMJ19 (BioWind). After the digestion product was recovered by gel, the target fragment and the vector were connected. The connection product was transformed into Escherichia coli DH5α competent cells, and the positive transformant was successfully identified and named pXMJ19-cipA.

1.2 The coding region DNA optimized according to the codon preference of Corynebacterium glutamicum was chemically synthesized based on the arginine deiminase (arc) gene sequence reported by Kim et al. (2007). The chemical synthesis was completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The arc gene sequence is as follows:

ATGAACAACGGCATCAACGTTAACTCCGAGATCGGCAAGCTGAAGTCCGTTCTGCTGCACCGCCCAGGCGCTGAGGTTGAGAACATCACCCCAGACACCATGAAGCAGCTGCTGTTCGACGACATCCCATACCTGAAGATCGCTCAGAAGGAGCACGACTTCTTCGCTCAGACCCTGCGCGACAACGGCGCTGAGACCGTTTACATCGAGAACCTGGCTACCGAGGTTTTCGAGAAGTCCTCCGAGACCAAGGAGGAGT TCCTGTCCCACCTGCTGCACGAGGCTGGCTACCGCCCAGGCCGCACCTA CGACGGCCTGACCGAGTACCTGACCTCCATGTCCACCAAGGACATGGTTGAGAAGATCTACGCTGGCGTTCGCAAGAACGAGCTGGACATCAAGCGCACCGCTCTGTCCGACATGGCTGGCTCCGACGCTGAGAACTACTTCTACCTGAACCCACTGCCAAACGCTTACTTCACCCGCGACCCACAGGCTTCCATGGGCGTTGGCATGACCATCAACAAGATGACCTTCCCAGCTCCAGCAGCCAGAGTCCCTGATCACCGAGT ACGTTATGGCTAACCACCCACGCTTCAAGGACACCCCAATCTGGC GCGACCGCAACCACCACCCGCATCGAGGGCGGCGACGAGCTGATCCTGAACAAGACCACCGTTGCTATCGGCGTTTCCGAGCGCACCTCCTCCAAGACCATCCAGAACCTGGCTAAGGAGCTGTTCGCTAACCCACTGTCCACCTTCGACACCGTTCTGGCTGTTGAGATCCCACACAACCACGCTATGATGCACCTGGACACCGTTTTCACCATGATCAACCACGACCAGTTCACCGTTTCCCAGGCATCATGGAC GGCGCTGGCAACATCAACGTTTTCATCCTGCGCCCAGGCAAGGACGAC GAGGTTGAGATCGAGCACCTGACCGACCTGAAGGCTGCTCTGAAGAAGGTTCTGAACCTGTCCGAGCTGGACCTGATCGAGTGCGGCGCTGGCGACCCAATCGCTGCTCCACGCGAGCAGTGGAACGACGGCTCCAACACCCTGGCTATCGCTCCAGGCGAGATCGTTACCTACGACCGCAACTACGTTACCGTTGAGCTGCTGAAGGAGCACGGCATCAAGGTTCACGAGATCCTGTCCTCCGAGCTGGGCC GCGGCCGCGGCGGCGCTCGCTGCATGTCCCAGCCACTGTGGCGCGAGGACCTGTAA

The synthesized gene sequence (SEQ ID NO.2) introduced an XhoI site at the 5' end of the DNA and a SacI site at the 3' end. After sequencing, the synthesized fragment was double-digested with XhoI/SacI, and the target gene and expression vector pXMJ19-cipA were connected after the digestion product was recovered by gel. The ligated product was transformed into Escherichia coli DH5α competent cells, and the positive transformant was successfully identified and named pXMJ19-cipA-arc, which was a genetically engineered bacterium containing arginine deiminase.

The genetically engineered bacteria containing arginine deiminase is deposited under the name of Corynebacterium glutamicum SUMHS-2020.01 and is classified as Corynebacterium glutamicum. The strain was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China on January 17, 2020. The deposit number of the culture collection center is CGMCC No.19404.

Example 2 Expression of fusion protein cipA-arc

2.1 Preparation of Corynebacterium glutamicum competent cells

Streak Corynebacterium glutamicum ATCC13032 on a plate containing LBG solid medium, culture in a 300C incubator, pick fresh strains and inoculate them in LBG liquid medium when the bacteria grow, and culture them in a shaking table at a temperature of 30°C and a speed of 200r/min for 12-24h. Transfer the activated bacterial solution to LBG medium at a 1% inoculum amount, and culture it in a shaking table at a temperature of 30°C and a speed of 200r/min until OD600 is about 0.9. Precool the bacterial solution in an ice-water mixture for 15-20min, then divide the precooled bacterial solution into sterilized 50mL centrifuge tubes in a clean bench, centrifuge at 4°C 6000g for 30s, and place the ice water for 2min. Aspirate the supernatant in the centrifuge tube, quickly add 2.5mL of precooled 10% glycerol to each centrifuge tube, and slowly blow and aspirate with a pipette until the bacteria are suspended. The suspension was centrifuged at 6000 g for 30 s at 4°C, the supernatant in the centrifuge tube was aspirated, 500 μL of pre-cooled 10% glycerol was quickly added to the centrifuge tube, and the cells were slowly pipetted until the bacteria were suspended. This operation was repeated three times to obtain competent cells of Corynebacterium glutamicum.

2.2 Electroporation of recombinant plasmid pXMJ19-cipA-arc into competent cells

Take 80μL competent cells and 2μL recombinant plasmid pXMJ19-cipA-arc, mix well, cool on ice for 10min; quickly add to ice-cold electric shock cup for electric shock, the electric shock conditions are voltage 2.5kV, time 5ms. After the pulse ends, remove the electric shock cup as soon as possible, add 1mL LBG liquid culture medium at room temperature, transfer to a centrifuge tube, gently shake and culture at 30℃ for 2h, take 200μL and spread on a 20μg/mL chloramphenicol resistance plate. Select a single colony to extract the plasmid, and then confirm the insertion of the target fragment by double enzyme digestion or PCR.

2.3 Induced expression of genetically engineered bacteria

The successfully identified recombinant bacteria were inoculated into LBG medium containing chloramphenicol at a final concentration of 20 μg/mL, the culture temperature was set at 30°C, the shaker speed was 200 r/min, and IPTG at a final concentration of 1 mM was added when the bacterial OD600 value reached 0.9, and the induction was carried out overnight at 30°C and 180 r/min. The whole cells of the recombinant bacteria were collected by centrifugation at 4°C, the bacteria were washed twice with 50 mM Tris-HCl at pH 7.0, and then resuspended in 50 mM phosphate buffer at pH 6.5. After ultrasonic disruption of the cells, the cells were centrifuged again at 4°C, and the precipitate was the obtained inclusion body protein cipA-arc (i.e., cipA-arc fusion protein).

2.4 Spectrophotometric determination of cipA-arc fusion protein activity

The enzymatic activity of the cipA-arginine deiminase fusion protein was determined by utilizing the specific colorimetric reaction of L-citrulline with diacetyl monoxime in a strongly acidic solution and the linear relationship between the absorbance of the reaction complex at 490 nm and the concentration of L-citrulline. Prepare substrate buffer (pH 6.0, 50 mM phosphate buffer) containing a final concentration of 200 mM [ ^14/15 N]-L-arginine, take 2.8 mL of substrate solution, add 0.2 mL of enzyme solution, and react at 37°C for 10 min. Dilute the enzyme reaction solution by an appropriate multiple (10-100 times), take 2 mL of the diluted reaction solution, add 3 mL of mixed acid (volume ratio _H2SO4 : _H3PO4 =1 _: 3) solution, 0.5 diacetyl- _oxime , semicarbazide mixture, shake well, immediately boil in a boiling water bath for 10 min, and measure the absorbance at 530 nm. Definition of cipA-arginine deiminase fusion protease activity: catalyzes [ ^14/15 The amount of enzyme that converts 1 μmol of citrulline from N]-L-arginine is defined as one unit of enzyme activity ((1U), and the activity of the fusion protease is 10000U. Specific enzyme activity is defined as the amount of enzyme activity contained in each mg of protein (U/mg). The protein concentration was determined by the Bradford method.

Example 3 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.1 is 20° C., the rotation speed during culture is 300 r/min, and the culture is carried out until the OD600 is about 0.3, the other experimental conditions and experimental steps are the same as those in Example 2.

Example 4 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.1 is 37° C., the rotation speed during culture is 150 r/min, and the culture is carried out until the OD600 is about 1.0, the other experimental conditions and experimental steps are the same as those in Example 2.

Example 5 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.1 is 40° C., the rotation speed during culture is 180 r/min, and the culture is carried out until the OD600 is about 0.72, the other experimental conditions and experimental steps are the same as those in Example 2.

Effects of different implementation conditions on the preparation of Corynebacterium glutamicum competent cells

Example Culture temperature (℃) Speed (r/min) OD ₆₀₀ Number of competent cells Example 2-2.1 30 200 0.9 normal Example 3 20 300 0.3 weak Example 4 37 150 1.0 normal Example 5 40 180 0.72 weak

Example 6 Expression of fusion protein cipA-arc

Except that the electric shock voltage in step 2.2 is 1 kV and the electric shock time is 10 ms, the other experimental conditions and experimental steps are the same as those in Example 2.

Example 7 Expression of fusion protein cipA-arc

Except that the voltage of the electric shock in step 2.2 is 5 kV and the duration of the electric shock is 1 ms, the other experimental conditions and experimental steps are the same as those in Example 2.

Effects of different electroporation conditions on transformation efficiency of Corynebacterium glutamicum competent cells

Example 8 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.3 is 30°C, the rotation speed during culture is 200 r/min, the culture is carried out until OD600 is about 0.9, the inducer concentration is 1.0 mM, the induction temperature is 30°C, and the rotation speed during induction is 180 r/min, the other experimental conditions and experimental steps are the same as those in Example 2.

Example 9 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.3 is 20°C, the rotation speed during culture is 300 r/min, the culture is carried out until OD600 is about 0.5, the inducer concentration is 0.5 mM, the induction temperature is 20°C, and the rotation speed during induction is 200 r/min, the other experimental conditions and experimental steps are the same as those in Example 2.

Example 10 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.3 is 25°C, the rotation speed during culture is 150 r/min, the culture is cultured until OD600 is about 1.0, the inducer concentration is 1.5 mM, the induction temperature is 35°C, and the rotation speed during induction is 220 r/min, the other experimental conditions and experimental steps are the same as Example 2.

Effects of different culture conditions on the expression efficiency of recombinant Corynebacterium glutamicum fusion protein cipA-arc

Example 11 Conversion of [ ^14/15 N]-L-arginine to produce [ ^14/15 N]-L-citrulline

Step 1: The immobilized fusion protein cipA-arc is suspended in a glass column reactor:

Take a plexiglass column with a height-to-diameter ratio of 15 as a filling column, and fill the immobilized fusion protein cipA-arc into the filling column.

Step 2: Synthesis of [ ^14/15 N]-L-citrulline catalyzed by immobilized fusion protein cipA-arc packed column:

Ammonium acetate buffer solution (0.2 mol/l, pH 6.0) with a molar concentration of [ ^14/15 N]-L-arginine of 2.5 mol/L was passed through the packed column at a flow rate of 0.3 BV/h at a temperature of 30°C. Under these reaction conditions, the conversion rate of [ ^14/15 N]-L-arginine was 99.8%, and each liter of fermentation broth contained 435 g of [ ^14/15 N]-L-citrulline. After 520 hours of conversion of the immobilized fusion protein cipA-arc, the catalytic activity of the fusion protein cipA-arc was still stable, that is, the service life of the immobilized fusion protein cipA-arc was 520 hours.

Step 3, separation and purification of [ ^14/15 N]-L-citrulline, comprises the following three steps:

a. Collect the reaction solution flowing out of the packed column, remove the buffer salt by nanofiltration, return it to the reaction system, recycle it, and collect the product concentrate;

b. The reaction solution after step a is concentrated and crystallized under vacuum to obtain [ ^14/15 N]-L-citrulline with a purity of more than 99.8%, which is then spray-dried to obtain a white powdery solid.

Example 12 Conversion of [ ^14/15 N]-L-arginine to produce [ ^14/15 N]-L-citrulline

Step 1: The immobilized fusion protein cipA-arc is suspended in a glass column reactor:

A plexiglass column with a height-to-diameter ratio of 25 was used as a filling column, and the immobilized fusion protein cipA-arc was filled into the filling column.

Step 2: Immobilized fusion protein cipA-arc packed column catalyzes the synthesis of L-citrulline:

The ammonium formate buffer solution (0.2 mol/L, pH 6.0) with a molar concentration of [ ^14/15 N]-L-arginine of 2.0 mol/L was passed through the packed column at a flow rate of 0.4 BV/h at a temperature of 35°C. Under these reaction conditions, the conversion rate of [ ^14/15 N]-L-arginine was 98.8%, and each liter of fermentation broth contained 348 g of [ ^14/15 N]-L-citrulline. After 560 hours of conversion of the immobilized fusion protein cipA-arc, the catalytic activity of the fusion protein cipA-arc was still stable, that is, the service life of the immobilized fusion protein cipA-arc was 560 hours.

Step 3, separation and purification of [ ^14/15 N]-L-citrulline, comprises the following three steps:

a. Collect the reaction solution flowing out of the packed column, remove the buffer salt by nanofiltration, return it to the reaction system, recycle it, and collect the product concentrate;

b. The reaction solution after step a is concentrated and crystallized under vacuum to obtain [ ^14/15 N]-L-citrulline with a purity of more than 99.5%, which is then spray-dried to obtain a white powdery solid.

Example 13 Conversion of [ ^14/15 N]-L-arginine to produce [ ^14/15 N]-L-citrulline

Step 1: Immobilize the fusion protein cipA-arc and suspend it in a glass column reactor:

A plexiglass column with a height-to-diameter ratio of 30 was used as a filling column, and the immobilized fusion protein cipA-arc was filled into the filling column.

Step 2: Synthesis of [ ^14/15 N]-L-citrulline catalyzed by immobilized fusion protein cipA-arc packed column:

An aqueous solution of ammonium chloride (0.3 mol/L, pH 4.5) with a molar concentration of [ ^14/15 N]-L-arginine of 1.5 mol/L was passed through the packed column at a flow rate of 0.5 BV/h at a temperature of 40°C. Under these reaction conditions, the conversion rate of [ ^14/15 N]-L-arginine was 99.5%, and each liter of fermentation broth contained 261 g of [ ^14/15 N]-L-citrulline. After 530 hours of conversion of the immobilized fusion protein cipA-arc, the catalytic activity of the fusion protein cipA-arc was still stable, that is, the service life of the immobilized fusion protein cipA-arc was 530 hours.

Step 3, separation and purification of [ ^14/15 N]-L-citrulline, comprises the following three steps:

a. Collect the reaction solution flowing out of the packed column, remove the buffer salt by nanofiltration, return it to the reaction system, recycle it, and collect the product concentrate;

b. The reaction solution after step a is concentrated and crystallized under vacuum to obtain [ ^14/15 N]-L-citrulline with a purity of more than 99.6%, which is then spray-dried to obtain a white powdery solid.

Example 14 Conversion of [ ^14/15 N]-L-arginine to produce [ ^14/15 N]-L-citrulline

Step 1: Immobilize the fusion protein cipA-arc and suspend it in a glass column reactor:

A plexiglass column with a height-to-diameter ratio of 40 was used as a filling column, and the immobilized fusion protein cipA-arc was filled into the filling column.

Step 2: Synthesis of [ ^14/15 N]-L-citrulline catalyzed by immobilized fusion protein cipA-arc packed column:

A 1.0 mol/L aqueous solution (pH 7.5) of [ ^14/15 N]-L-arginine was passed through a packed column at a flow rate of 0.3 BV/h at a temperature of 55°C. Under these reaction conditions, the conversion rate of [ ^14/15 N]-L-arginine was 95%, and each liter of fermentation broth contained 174 g of [ ^14/15 N]-L-citrulline. After 480 hours of conversion of the immobilized fusion protein cipA-arc, the catalytic activity of the fusion protein cipA-arc was still stable, that is, the service life of the immobilized fusion protein cipA-arc was 480 hours.

Step 3, separation and purification of [ ^14/15 N]-L-citrulline, comprises the following three steps:

a. Collect the reaction solution flowing out of the packed column, remove the buffer salt by nanofiltration, return it to the reaction system, recycle it, and collect the product concentrate;

b. The reaction solution after step a is concentrated and crystallized under vacuum to obtain [ ^14/15 N]-L-citrulline with a purity of more than 99.5%, which is then spray-dried to obtain a white powdery solid.

Example 15 Conversion of [ ^14/15 N]-L-arginine to produce [ ^14/15 N]-L-citrulline

Step 1: Immobilize the fusion protein cipA-arc and suspend it in a glass column reactor:

Take a plexiglass column with a height-to-diameter ratio of 35 as a filling column, and fill the immobilized fusion protein cipA-arc into the filling column.

Step 2: Synthesis of [ ^14/15 N]-L-citrulline catalyzed by immobilized fusion protein cipA-arc packed column:

An aqueous solution of ammonium bicarbonate (0.3 mol/L, pH 8.5) with a molar concentration of [ ^14/15 N]-L-arginine of 1.8 mol/l was passed through the packed column at a flow rate of 0.3 BV/h at a temperature of 20°C. Under these reaction conditions, the conversion rate of [ ^14/15 N]-L-arginine was 98%, and each liter of fermentation broth contained 313 g of [ ^14/15 N]-L-citrulline. After 450 hours of conversion of the immobilized fusion protein cipA-arc, the catalytic activity of the fusion protein cipA-arc was still stable, that is, the service life of the immobilized fusion protein cipA-arc was 450 hours.

Step 3, separation and purification of [ ^14/15 N]-L-citrulline, comprises the following three steps:

a. Collect the reaction solution flowing out of the packed column, remove the buffer salt by nanofiltration, return it to the reaction system, recycle it, and collect the product concentrate;

b. The reaction solution after step a is concentrated and crystallized under vacuum to obtain [ ^14/15 N]-L-citrulline with a purity of more than 99.7%, which is then spray-dried to obtain a white powdery solid.

The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical contents disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

SEQUENCE LISTING

<110> Shanghai University of Medicine and Health Sciences

<120> A method for continuously preparing [14/15N]-L-citrulline using an immobilized enzyme

<130> 2019.11.6

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 312

<212> PRT

<213> Artificial sequence

<400> 1

ATGATCAACGACATGCACCCATCCCTGATCAAGGACAAGGACATGATGGACGACGTTATGCTGCGCTCCTGCAAGATCATCGCTATGAAGATCATGCCAGACAAGGTTATGCAGGTTATGGTTACCGTTCTGATGCTGGACGGCACCTCCGAGGAGATGCTGCTGAAGTGGAACCTGCTGGACAACCGCGGCATGGCTATCTACAAGGTTCTGATGGAGGCTCTGTGCGGCAAGAAGGACGTTTAAGATCGGCACCGTT GGCAAGGTTGGCCCACTGGGCTGCGACTACATCAACTGCGTTGAGATCTCCATG

<210> 2

<211> 1233

<212> PRT

<213> Artificial sequence

<400> 2

ATGAACAACGGCATCAACGTTAACTCCGAGATCGGCAAGCTGAAGTCCGTTCTGCTGCACCGCCCAGGCGCTGAGGTTGAGAACATCACCCCAGACACCATGAAGCAGCTGCTGTTCGACGACATCCCATACCTGAAGATCGCTCAGAAGGAGCACGACTTCTTCGCTCAGACCCTGCGCGACAACGGCGCTGAGACCGTTTACATCGAGAACCTGGCTACCGAGGTTTTCGAGAAGTCCTCCGAGACCAAGGAGGAGT TCCTGTCCCACCTGCTGCACGAGGCTGGCTACCGCCCAGGCCGCACCTA CGACGGCCTGACCGAGTACCTGACCTCCATGTCCACCAAGGACATGGTTGAGAAGATCTACGCTGGCGTTCGCAAGAACGAGCTGGACATCAAGCGCACCGCTCTGTCCGACATGGCTGGCTCCGACGCTGAGAACTACTTCTACCTGAACCCACTGCCAAACGCTTACTTCACCCGCGACCCACAGGCTTCCATGGGCGTTGGCATGACCATCAACAAGATGACCTTCCCAGCTCCAGCAGCCAGAGTCCCTGATCACCGAGT ACGTTATGGCTAACCACCCACGCTTCAAGGACACCCCAATCTGGC GCGACCGCAACCACCACCCGCATCGAGGGCGGCGACGAGCTGATCCTGAACAAGACCACCGTTGCTATCGGCGTTTCCGAGCGCACCTCCTCCAAGACCATCCAGAACCTGGCTAAGGAGCTGTTCGCTAACCCACTGTCCACCTTCGACACCGTTCTGGCTGTTGAGATCCCACACAACCACGCTATGATGCACCTGGACACCGTTTTCACCATGATCAACCACGACCAGTTCACCGTTTCCCAGGCATCATGGAC GGCGCTGGCAACATCAACGTTTTCATCCTGCGCCCAGGCAAGGACGAC GAGGTTGAGATCGAGCACCTGACCGACCTGAAGGCTGCTCTGAAGAAGGTTCTGAACCTGTCCGAGCTGGACCTGATCGAGTGCGGCGCTGGCGACCCAATCGCTGCTCCACGCGAGCAGTGGAACGACGGCTCCAACACCCTGGCTATCGCTCCAGGCGAGATCGTTACCTACGACCGCAACTACGTTACCGTTGAGCTGCTGAAGGAGCACGGCATCAAGGTTCACGAGATCCTGTCCTCCGAGCTGGGCC GCGGCCGCGGCGGCGCTCGCTGCATGTCCCAGCCACTGTGGCGCGAGGACCTGTAA

Claims (10)

1. a kind of method that immobilized enzyme prepares [ ^14/15N ]-L-citrulline continuously, is characterized in that, described method comprises the steps: (1) suspending the fusion protein comprising immobilized enzyme in a packed bed reactor; The fusion protein containing the immobilized enzyme is the inclusion body protein cipA-arc with catalytic activity obtained by immobilizing arginine deiminase arc on the inclusion body protein cipA by using cipA as a carrier, that is, the cipA-arc fusion protein, The sequence of cipA is SEQ ID NO.1, the sequence of arginine deiminase arc is SEQ ID NO.2, and the cipA-arc fusion protein is prepared by the following steps: S1 preparation of Corynebacterium glutamicum competent cells; S2 Using the recombinant plasmid pXMJ19-cipA-arc to transform the Corynebacterium glutamicum competent cells described in step S1 by electroporation to obtain recombinant bacteria; S3: After the obtained recombinant bacteria are induced and expressed by the genetically engineered bacteria, the bacterial cells are ultrasonically disrupted and centrifuged, and the resulting precipitate is the cipA-arc fusion protein; (2) React the solution containing [ ^14/15 N]-L-arginine at a flow rate of 0.3-0.5BV/h through a packed bed reactor at 20-55°C, and the reaction solution is separated and purified [ ^14/15 N]-L-citrulline can be obtained. 2. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, described Corynebacterium glutamicum competent cell adopts following preparation method: After culturing Corynebacterium glutamicum ATCC13032 in LB-containing solid medium, pick fresh strains and inoculate them in LB liquid medium, and transfer the activated bacterial solution to LB medium at an inoculation amount of 0.8-1.5% after cultivation , continue to cultivate until the OD600 is 0.8-1.0; pre-cool the bacterial solution with ice-water mixture, centrifuge, suck out the supernatant, add glycerin, blow and suck until the bacteria are suspended, centrifuge again, suck out the supernatant, and then add glycerol. Blow and suck until the bacteria are suspended, and then the competent cells of Corynebacterium glutamicum can be obtained. 3. the method for the continuous preparation of [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, described recombinant bacterium is preserved in China Microorganism Strain Preservation Management Committee General Microorganism Center, The deposit number is CGMCCNo.19404. 4. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 2, is characterized in that, described recombinant plasmid pXMJ19-cipA-arc electric shock transforms competent cell and adopts following preparation method: Take the competent cells of Corynebacterium glutamicum and the recombinant plasmid pXMJ19-cipA-arc, mix well, cool on ice, under the same temperature conditions, electric shock condition is 1-5kV, electric shock 1-10ms; then add LB medium, transferred to a centrifuge tube, after shaking culture, the obtained liquid was spread on a chloramphenicol-resistant plate, a single colony was selected to extract the plasmid, and then the insertion of the target fragment was confirmed by double enzyme digestion and PCR, and the obtained recombinant bacteria inoculation. 5. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 3, is characterized in that, the induction expression method of described genetically engineered bacteria is as follows: The recombinant bacteria were inoculated in LB medium containing chloramphenicol, cultured on a shaker until the OD600 value of the bacteria reached 0.8-1.0, adding isopropyl-β-D-thiogalactoside, and collected by centrifugation after overnight induction The recombinant bacterial cells were washed with Tris-HCl buffer, resuspended in phosphate buffer, ultrasonically disrupted, and then centrifuged again to obtain the cipA-arc fusion protein. 6. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, described recombinant plasmid pXMJ19-cipA-arc is prepared by the following method: (1) The cipA gene whose sequence is SEQ ID NO.1 is introduced into the HindIII site at the 5' end of the DNA, and the SalI site is introduced at the 3' end, and the synthesized fragments are sequenced, and the target gene and the expression vector are digested with HindIII/SalI double enzymes pXMJ19, after the digested product was recovered by gel, the target fragment was ligated with the vector, and the ligated product was transformed into Escherichia coli DH5α competent cells to obtain the positive transformant expression vector pXMJ19-cipA; (2) The arginine deiminase arc gene whose sequence is SEQ ID NO.2 is introduced into the XhoI site at the 5' end of the DNA, and the SacI site is introduced at the 3' end. Digest the target gene and the expression vector pXMJ19-cipA. After the digested product is recovered by gel, the target fragment and the vector are ligated, and the ligated product is transformed into E. coli DH5α competent cells to obtain the positive transformant recombinant plasmid pXMJ19-cipA-arc. It is a genetically engineered bacterium containing arginine deiminase. 7. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, expresses in the genetically engineered bacterium containing arginine deiminase Arginine deiminase. 8. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that the fusion protease activity comprising immobilized enzyme described in step (1) is 9000 -12000U, the concentration of [ ^14/15 N]-L-arginine in the solution containing [ ^14/15 N]-L-arginine in the step (2) is 1.0-2.5mol/L. 9. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, the solution comprising [ ^14/15 N]-L-arginine Any one of ammonium acetate buffer solution, ammonium formate buffer solution, ammonium chloride aqueous solution, ammonium bicarbonate aqueous solution or pure aqueous solution is also included. 10. the method for continuously preparing [ ^14/15 N]-L-citrulline by immobilized enzyme according to claim 1, is characterized in that, the solution comprising [ ^14/15 N]-L-arginine Any one of ammonium acetate buffer solution, ammonium formate buffer solution, ammonium chloride aqueous solution or ammonium bicarbonate aqueous solution is also included.