阅读说明：本技术 一种固定化酶连续制备[14/15n]-l-瓜氨酸的方法 (Continuous preparation of immobilized enzyme14/15N]Method for producing L-citrulline ) 是由 黄钢 李斌 于 2020-04-23 设计创作，主要内容包括：本申请公开了一种固定化酶连续制备[<Sup>14/</Sup><Sup>15</Sup>N]-L-瓜氨酸的方法,属于酶学与酶工程技术领域。其方法是将包含固定化酶的融合蛋白悬浮于填充床反应器；再将包含[<Sup>14/15</Sup>N]-L-精氨酸的溶液在20-55℃条件下,以流速0.3-0.5BV/h流经填充床反应器进行反应,反应液经分离、纯化即可得到[14/15N]-L-瓜氨酸。本发明的技术构思是采用cipA固定化融合蛋白为载体将精氨酸脱亚胺酶arc固定于包涵体蛋白cipA上,产生具有催化活性的包涵体蛋白cipA-arc,即cipA-arc融合蛋白。本申请提供的具有催化活性的固定化cipA-精氨酸脱亚胺酶cipA-arc融合蛋白,能连续反应560小时以上；同时得到的具有同位素标记的[<Sup>14/15</Sup>N]L-瓜氨酸,为前列腺疾病、心血管疾病等的诊断与治疗提供了有效途径。(The application discloses a continuous preparation of immobilized enzyme 14/ 15 N]A method of-L-citrulline, belonging to the technical field of enzymology and enzyme engineering. The method is that the fusion protein containing the immobilized enzyme is suspended in a packed bed reactor; then will comprise 14/15 N]The solution of-L-arginine flows through a packed bed reactor at the flow rate of 0.3-0.5BV/h for reaction at the temperature of 20-55 ℃, and the reaction solution is separated and purified to obtain the [14/15N]-L-citrulline. The invention adopts the technical conception that the cipA immobilized fusion protein is used as a carrier to fix arginine deiminase arc on the inclusion body protein cipA, and the inclusion body protein cipA-arc with catalytic activity, namely the cipA-arc fusion protein, is generated. The application provides an immobilized cipA-arginine deiminase cipA-arc fusion protein with catalytic activityWhite, and can continuously react for more than 560 hours; simultaneously obtaining the product with isotope labeling 14/15 N]The L-citrulline provides an effective way for diagnosing and treating prostate diseases, cardiovascular diseases and the like.)

1. Continuous preparation of immobilized enzymePreparation [ 2 ]^14/15N]-L-citrulline, characterized in that it comprises the following steps:

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

(2) will comprise^14/15N]The solution of the L-arginine flows through a packed bed reactor at the flow rate of 0.3 to 0.5BV/h for reaction at the temperature of between 20 and 55 ℃, and the reaction solution is separated and purified to obtain the product^14/15N]-L-citrulline.

2. The immobilized enzyme of claim 1, which is continuously produced^14/15N]-L-citrulline, characterized in that said immobilized enzyme-containing fusion protein in step (1) is a catalytically active inclusion body protein cipA-arc obtained by immobilizing arginine deiminase arc on inclusion body protein cipA using cipA as a carrier, i.e. a cipA-arc fusion protein, said cipA-arc fusion protein being prepared by the steps of:

(1) preparing corynebacterium glutamicum competent cells;

(2) transforming the corynebacterium glutamicum competent cell in the step (1) by adopting recombinant plasmid pXMJ19-cipA-arc electric shock to obtain a recombinant thallus whole cell;

(3) and (3) carrying out ultrasonic crushing and centrifugation on the recombinant bacteria obtained in the step (2) to obtain a recombinant bacteria whole cell through the induction expression of the genetic engineering bacteria, and obtaining a precipitate, namely the cipA-arc fusion protein.

3. The immobilized enzyme of claim 2, which is continuously produced^14/15N]-L-citrulline, characterized in that said c.glutamicum competent cells are prepared as follows:

culturing Corynebacterium glutamicum ATCC13032 in a LBG-containing solid culture medium, selecting a fresh strain, inoculating the fresh strain into an LBG liquid culture medium, culturing, transferring an activated bacterial liquid into the LBG culture medium according to the inoculum size of 0.8-1.5%, and continuously culturing until OD600 is 0.8-1.0; precooling and centrifuging the bacterial liquid by using an ice-water mixture, sucking out supernatant, adding glycerol, blowing and sucking until thalli are suspended, centrifuging again, sucking out supernatant, adding glycerol, blowing and sucking until thalli are suspended, and thus obtaining the corynebacterium glutamicum competent cells.

4. The immobilized enzyme of claim 3, which is continuously produced^14/15N]-method for L-citrulline, characterized in that said recombinant plasmid pXMJ19-cipA-arc shock transformed competent cells are prepared as follows:

uniformly mixing the corynebacterium glutamicum competent cells and the recombinant plasmid pXMJ19-cipA-arc, cooling on ice, and performing electric shock for 1-10ms under the same temperature condition with the voltage of 1-5 kV; adding an LBG liquid culture medium at room temperature, transferring the liquid culture medium into a centrifuge tube, carrying out shake culture, taking the obtained liquid, coating the liquid on a chloramphenicol-resistant plate, selecting a single colony to extract a plasmid, confirming the insertion of a target fragment through double enzyme digestion and PCR, and inoculating the obtained recombinant bacteria.

5. The immobilized enzyme of claim 4, which is continuously produced^14/15N]The method for inducing and expressing the L-citrulline is characterized in that the method for inducing and expressing the genetically engineered bacteria comprises the following steps:

inoculating the recombinant bacteria into an LBG culture medium containing chloramphenicol, adding isopropyl-beta-D-thiogalactoside when the OD600 value of the bacteria reaches 0.8-1.0 through shaking table culture, centrifugally collecting the whole cells of the recombinant bacteria after induction overnight, washing the bacteria with Tris-HCl buffer solution, then re-suspending in phosphate buffer solution, ultrasonically breaking the cells, then centrifuging again, and precipitating to obtain the cipA-arc fusion protein.

6. The immobilized enzyme of claim 2, which is continuously produced^14/15N]-method for L-citrulline, characterized in that said recombinant plasmid pXMJ19-cipA-arc is prepared by the following method:

(1) introducing a HindIII site into a CipA gene sequence at the end of DNA5 ', introducing a SalI site into the end of 3' to obtain a fragment with the gene sequence of SEQ ID NO.1, sequencing the synthesized fragment, performing double enzyme digestion on a target gene and an expression vector pXMJ19 by HindIII/SalI, recovering an enzyme digestion product through gel, connecting the target fragment and the vector, and transforming escherichia coli DH5 alpha competent cells by the connection product to obtain a positive transformant expression vector pXMJ 19-cipA;

(2) introducing an XhoI site into an arginine deiminase arc gene sequence at the end of DNA5 ', introducing a SacI site into the end of 3' to obtain a fragment with a gene sequence of SEQ ID NO.2, sequencing the synthesized fragment, performing double digestion on a target gene and an expression vector pXMJ19-cipA by using XhoI/SacI, recovering a digestion product through gel, connecting the target fragment and the vector, transforming escherichia coli DH5 alpha competent cells by using the connection product, and obtaining a positive transformant recombinant plasmid pXMJ19-cipA-arc, namely the genetic engineering bacterium containing the arginine deiminase.

7. The immobilized enzyme of claim 6, which is continuously produced^14/15N]The method for preparing L-citrulline is characterized in that the preservation name of the genetically engineered bacteria containing arginine deiminase is Corynebacterium glutamicum SUMHS-2020.01, the genetically engineered bacteria are classified and named Corynebacterium glutamicum, the strain is preserved in China general microbiological culture Collection center of China institute of microbiology, No.1 Hokkaido, No. 3 Hokkaido, North Asia City, Beijing, China, 1 month and 17 days of 2020, and the preservation number of the strain preservation center is CGMCC No. 19404.

8. The immobilized enzyme of claim 7, which is continuously produced^14/15N]The method for producing L-citrulline is characterized in that the arginine deiminase is expressed in the genetic engineering bacteria containing the arginine deiminase.

9. The immobilized enzyme of claim 1, which is continuously produced^14/15N]The process of (E) -L-citrulline, characterized in that the activity of the fusion protease comprising the immobilized enzyme in step (1) is 9000-^14/15N]In a solution of (E) -L-arginine^14/15N]The concentration of the-L-arginine is 1.0-2.5 mol/L.

10. The immobilized enzyme of claim 1, which is continuously produced^14/15N]-L-citrulline, characterized in thatThe composition comprises^14/15N]-the solution of L-arginine further comprises any one of a buffered solution of ammonium acetate, a buffered solution of ammonium formate, an aqueous solution of ammonium chloride, an aqueous solution of ammonium bicarbonate or a pure aqueous solution;

preferably, the composition comprises^14/15N]The solution of L-arginine further comprises any one of a buffered solution of ammonium acetate, a buffered solution of ammonium formate, an aqueous solution of ammonium chloride or an aqueous solution of ammonium bicarbonate.

Technical Field

The application relates to a method for producing high-purity [ alpha ]^14/15N]A method of decomposing (E) -L-citrulline, particularly a method of decomposing (E) by using a recombinant arginine deiminase^14/15N]Production of high-purity [ alpha ], [ alpha ] -L-arginine^14/15N]A method of-L-citrulline, belonging to the technical field of enzymology and enzyme engineering.

Background

L-citrulline (L-citrulline) is a specific amino acid. Participate in various metabolic processes in vivo, such as free radical removal, foreign body rejection effect indicators, vasodilation, blood pressure stabilization, rheumatoid arthritis diagnosis, oxidation resistance and the like, prevent prostate diseases, improve sexual functions, resist aging, enhance immunity and the like, and have very wide application prospects.

The method for producing L-citrulline comprises the following steps: chemical, fermentation, enzymatic methods. The chemical method is to hydrolyze L-arginine under alkaline condition to obtain L-citrulline, the control of the hydrolysis process is difficult, the product contains optical antipode D-citrulline, the product quality is influenced, and a large amount of waste water is generated in the production process to pollute the environment; the difficulty of the fermentation production is that the yield of the L-citrulline in unit volume is low, the operation process for extracting the L-citrulline from the fermentation liquor is complex, the yield is low, and the cost is high; the enzyme production refers to that under the action of arginine deiminase, L-arginine is converted into L-citrulline, and the method has the advantages of strong specificity and high product concentration, but the method also has the defect of low catalyst utilization rate, namely, thallus is prepared by re-fermentation after each secondary delivery, so that a large amount of raw materials are consumed, a large amount of wastewater is generated, and the environmental protection treatment cost is increased; and impurities (a large amount of bacteria, protein, various metal ions and other impurities remained in the fermentation liquor) brought into a reaction system by an enzyme catalyst, so that in the L-citrulline post-treatment process, the product is separated and purified by a series of process steps of removing bacteria, removing protein, separating and exchanging columns and the like, and the production cost is further increased.

The chemical method, the fermentation method or the enzymatic method for producing the L-citrulline have a plurality of defects, so that the production cost is high, and great difficulty is brought to practical popularization and application.

In order to overcome the problems, a solution of immobilized enzymes or cells is proposed, and the preparation methods of the immobilized enzymes or cells include two major types, namely physical methods and chemical methods. The physical methods include physical adsorption, entrapment and the like. The physical method for fixing the enzyme has the advantages that the enzyme does not participate in chemical reaction, the whole structure is kept unchanged, and the catalytic activity of the enzyme is well reserved. However, since the inclusion or semi-permeable membrane has a steric or steric hindrance, it is not suitable for some reactions. The chemical method includes a binding method and a crosslinking method. The combination between the chemical enzyme and the carrier is tight, the chemical enzyme is not easy to fall off, the stability is good, but the reaction condition is violent, the operation is complex, the control condition is harsh, and the activity loss is large.

Continuous preparation of L-citrulline [ J ] from Zhengpu (bright day, Zhang Qi.) in 2008 by immobilized pseudomonas cells in packed bed reactor]Food and biotechnology newspaper, 2008, 27 (5): 1673-^-1The operation is continuously carried out for 54d under the condition of (gram citrulline produced by each gram of cells per hour), but the fermentation production process of the thalli is complex, the time of immobilized cells is too long, the concentration of a substrate is low, the yield is not high, the immobilized cells are simple, but the problems of cell body breakage and release of hybrid protein and other organic substances in the thalli still exist, the difficulty of separation and purification of products in a reaction system is increased, the step of operation is also increased in cell immobilization, and the production cost is increased.

Disclosure of Invention

According to a first aspect of the present application, there is provided a continuous preparation of an immobilized enzyme^14/15N]-L-citrulline, said method comprising the steps of:

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

(2) will comprise^14/15N]The solution of the L-arginine flows through a packed bed reactor at the temperature of 20-55 ℃ and the flow rate of 0.3-0.5 BV/h.

Alternatively, the fusion protein comprising an immobilized enzyme in step (1) is 9000-12000U, preferably 10000U, and the fusion protein comprising an immobilized enzyme isThe step (2) comprises^14/15N]In a solution of (E) -L-arginine^14/15N]The concentration of the-L-arginine is 1.0-2.5 mol/L.

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

Optionally, the composition comprises^14/15N]In a solution of (E) -L-arginine^14/15N]The concentration of the-L-arginine is 1.0-2.5 mol/L.

Optionally, the fusion protein containing the immobilized enzyme in step (1) is inclusion body protein cipA-arc obtained by immobilizing arginine deiminase arc on inclusion body protein cipA by using cipA as a carrier, namely, the fusion protein of cipA-arc.

Alternatively, the cipA-arc fusion protein is prepared by:

(1) preparing corynebacterium glutamicum competent cells;

(2) transforming the corynebacterium glutamicum competent cells in the step (1) by adopting a recombinant plasmid pXMJ19-cipA-arc electric shock to obtain a recombinant strain;

(3) and (3) carrying out ultrasonic crushing and centrifugation on the recombinant bacteria obtained in the step (2) to obtain a recombinant bacteria whole cell through the induction expression of the genetic engineering bacteria, and obtaining a precipitate, namely the cipA-arc fusion protein.

Alternatively, the corynebacterium glutamicum competent cell is prepared by the following method:

culturing Corynebacterium glutamicum ATCC13032 in a LBG-containing solid culture medium, selecting a fresh strain, inoculating the fresh strain into an LBG liquid culture medium, culturing, transferring an activated bacterial liquid into the LBG culture medium according to the inoculum size of 0.8-1.5%, and continuously culturing until OD600 is 0.8-1.0; precooling and centrifuging the bacterial liquid by using an ice-water mixture, sucking out supernatant, adding glycerol, blowing and sucking until thalli are suspended, centrifuging again, sucking out supernatant, adding glycerol, blowing and sucking until thalli are suspended, and thus obtaining the corynebacterium glutamicum competent cells.

Preferably, the Corynebacterium glutamicum ATCC13032 is streaked on a plate containing LBG solid culture medium, cultured in an incubator, a fresh strain is picked up after thalli grow out and inoculated in an LBG liquid culture medium, and cultured for 12-24h in a shaking table with the temperature of 20-40 ℃ and the rotating speed of 150-300 r/min; transferring the activated bacteria liquid to LBG culture medium according to 1% inoculation amount, and culturing in a shaking table at the temperature of 20-40 ℃ and the rotating speed of 150-; placing the bacterial liquid in an ice water mixture for precooling for 15-20min, subpackaging the precooled bacterial liquid in a super clean bench into sterilized centrifuge tubes, centrifuging at 6000g at 4 ℃ for 30s, and placing ice water for 2 min; sucking out the supernatant in the centrifuge tube, adding precooled 10% glycerol into the centrifuge tube respectively, and slowly sucking the mixture by using a pipette gun until the bacteria are suspended; the suspension is centrifuged at 6000g for 30s at 4 ℃, the supernatant in the centrifuge tube is sucked out, precooled 10% glycerol is added into the centrifuge tube, and the suspension is slowly sucked by a pipette gun until the thalli are suspended.

Optionally, the temperature of the culture is 30 ℃; the rotating speed during the culture is 200 r/min.

Alternatively, the recombinant plasmid pXMJ19-cipA-arc shock-transformed competent cells were prepared by the following method:

uniformly mixing the corynebacterium glutamicum competent cells and the recombinant plasmid pXMJ19-cipA-arc, cooling on ice, and performing electric shock for 1-10ms under the same temperature condition with the voltage of 1-5 kV; adding an LBG liquid culture medium at room temperature, transferring the liquid culture medium into a centrifuge tube, carrying out shake culture, taking the obtained liquid, coating the liquid on a chloramphenicol-resistant plate, selecting a single colony to extract a plasmid, confirming the insertion of a target fragment through double enzyme digestion and PCR, and inoculating the obtained recombinant bacteria.

Taking the competent cells and the recombinant plasmid pXMJ19-cipA-arc as a preferable scheme, mixing uniformly, and cooling for 10min on ice; rapidly adding an ice-cold electric shock cup for electric shock, wherein the electric shock condition is that the voltage is 2-4kV, and the time is 3-7 ms; taking out the electric shock cup as soon as possible after the pulse is finished, adding the LBG liquid culture medium at room temperature, transferring the electric shock cup into a centrifuge tube, carrying out soft shaking culture for 2 hours, and coating the obtained liquid on a chloramphenicol resistant plate containing 20 mu g/mL; and selecting a single colony to extract a plasmid, and confirming the insertion of the target fragment by double enzyme digestion or PCR.

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

Optionally, the method for inducing expression of the genetically engineered bacteria is as follows:

inoculating the recombinant bacteria into an LBG culture medium containing chloramphenicol, adding isopropyl-beta-D-thiogalactoside when the OD600 value of the bacteria reaches 0.8-1.0 through shaking table culture, centrifugally collecting the whole cells of the recombinant bacteria after induction overnight, washing the bacteria with Tris-HCl buffer solution, then re-suspending in phosphate buffer solution, ultrasonically breaking the cells, then centrifuging again, and precipitating to obtain the cipA-arc fusion protein.

Preferably, the successfully identified recombinant bacteria are inoculated into an LBG culture medium containing chloramphenicol with the final concentration of 20 mu g/mL, the culture temperature is set to be 20-40 ℃, the rotating speed of a shaking table is 150-; and (3) centrifugally collecting recombinant bacterial cells at 4 ℃, washing the bacterial cells by using a buffer solution, then suspending the bacterial cells in another buffer solution, ultrasonically breaking the cells, then centrifugally collecting the cells at 4 ℃, and precipitating to obtain the cipA-arc fusion protein.

Optionally, the temperature of both the culturing and the inducing is 30 ℃; the rotating speed during the culture is 200 r/min; the rotating speed during induction is 180 r/min.

Optionally, the wash buffer is Tris-HCl; the other resuspended buffer is phosphate buffer;

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

preferably, the phosphate buffer has a pH of 6.5.

Optionally, the genetically engineered bacteria express arginine deiminase.

Optionally, the genetically engineered bacterium is constructed by the following method:

1) introducing a HindIII site into a cipA gene sequence at the end of DNA5 ', introducing a SalI site into the end of 3' to obtain a fragment with a gene sequence of SEQ ID NO.1, sequencing the synthesized fragment, performing double enzyme digestion on a target gene and an expression vector pXMJ19 by HindIII/SalI, recovering a digestion product through gel, connecting the target fragment and the vector, and transforming escherichia coli DH5 alpha competent cells by the connection product to obtain a positive transformant pXMJ 19-cipA;

2) introducing an arc gene sequence into an XhoI site at the end of DNA5 ', introducing a SacI site at the end of 3' to obtain a fragment with a gene sequence of SEQ ID NO.2, sequencing the synthesized fragment, performing double enzyme digestion on a target gene and an expression vector pXMJ19-cipA by using XhoI/SacI, recovering an enzyme digestion product through gel, connecting the target fragment and the vector, and transforming a Escherichia coli DH5 alpha competent cell by using the connection product to obtain a positive transformant pXMJ19-cipA-arc, namely the genetic engineering bacteria containing arginine deiminase.

The genetically engineered bacterium containing arginine deiminase is preserved as Corynebacterium glutamicum SUMHS-2020.01, and is classified and named as Corynebacterium glutamicum, and the strain is preserved in China general microbiological culture Collection center of China institute of microbiology, national academy of sciences, China, institute of microbiology, No.1, 3, of the south China area of the West Lu, No.1, of the Chaoyang district, in 2020, 1 month and 17 days, and the preservation number of the strain preservation center is CGMCC No. 19404.

The invention also provides a technical scheme for expressing the arginine deiminase in the genetic engineering bacteria containing the arginine deiminase.

Optionally, the composition comprises^14/15N]The solution of L-arginine also comprises a buffered solution of ammonium acetate, a buffered solution of ammonium formate, an aqueous solution of ammonium chloride, an aqueous solution of ammonium bicarbonate or an aqueous solution.

Preferably, the composition comprises^14/15N]The solution of L-arginine also comprises a buffer solution of ammonium acetate, ammonium formate, aqueous ammonium chloride or aqueous ammonium bicarbonate.

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

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

Optionally, the separation and purification steps are as follows:

1) collecting reaction liquid flowing out of the packed bed reactor, removing buffer salt through nanofiltration, returning to a reaction system for recycling, and collecting a product concentrated solution;

2) vacuum concentrating, crystallizing and drying the reaction solution obtained in the step 1) to obtain white powdery solid with the purity of more than 99.5%^14/15N]-L-citrulline.

In the present application, the "cipA gene sequence" refers To the sequence of the cipA gene reported by Kirsten Jung et al (Wang Y, Heermann R, JungK. CipA and CipB as scans folds To organic Proteins into crystals Inclusions [ J ]. ACS Synthetic Biology,2017,6,826 one 836).

In the present application, the term "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 arc deiminase from Lactococcus lactis ssp. lactis ATCC 7962in Escherichia coli BL21[ J ]. protein expression and Purification,2007,53(1): 0-15).

In the present application, "pXMJ 19" refers to a vector carrying a gene expression protein in corynebacterium glutamicum.

The beneficial effects that this application can produce include:

1) the gene of the arginine deiminase is synthesized and cloned, a high-yield arginine deiminase engineering bacterium is constructed, and the arginine deiminase is expressed in corynebacterium glutamicum;

2) the application fixes the arginine deiminase on the inclusion body protein cipA to generate the inclusion body protein cipA-arc (namely the cipA-arc fusion protein) with catalytic activity, and the immobilization mode is simple and quick, low in cost, high in efficiency and convenient to use;

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

4) the inclusion body protein cipA-arc, arc-cipA immobilized fusion protein provided by the application can catalyze [ alpha ], [ alpha ] -alpha-amino acid^14/15N]Conversion of (E) -L-arginine to (2)^14/15N]The L-citrulline is prepared by a fixed bed reactor, has simple post-treatment, convenient product separation and purification, low cost and easy scale-up production, and is prepared by an enzyme method^14/15N]-L-citrullineA new way is added to acid;

5) the isotopically labeled [ alpha ], [^14/15N]The L-citrulline provides an effective way for diagnosing and treating prostate diseases, cardiovascular diseases and the like.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise indicated, the materials in the examples of the present application were obtained from the discovery platform by purchase of plasmid pXMJ19 from Wuhan vast Ling Biotech, Inc.

Corynebacterium glutamicum ATCC13032 was purchased from the Collection of microorganisms of Guangdong province.

[^14/15N]-L-citrulline assay method: the product [ 2 ] was measured by HPLC^14/15N]L-citrulline, a chromatographic column C18, 5 μm, 250mm × 4.6.6 mm, a mobile phase of 5% methanol, a flow rate of 1mL/min, a detection wavelength of 290nm, and a column temperature of room temperature.

The enzyme activity of the cipA-arginine deiminase is defined as catalysis [ alpha ] per minute at 37 ℃ and pH6.0^14/15N]Conversion of the-L-arginine to 1. mu. mol of^14/15N]The enzyme amount of L-citrulline is defined as one unit of enzyme activity ((1U).

Specific enzyme activity definition: the amount of enzyme activity per mg of protein (U/mg). Protein concentration was determined by the Bradford method.

According to an embodiment of the application, mainly include: 1) chemically synthesizing a target gene (cipA, arc); 2) continuously connecting the synthesized cipA and arc with a vector pXMG19 to construct an expression vector pXMJ 19-cipA-arc; 3) introducing pXMJ19-cipA-arc into Corynebacterium glutamicum ATCC13032 by electrotransformation; 4) inducing expression and separating inclusion body protein cipA-arc (namely, cipA-arc fusion protein); 5) the inclusion body protein cipA-arc is utilized to continuously catalyze the arginine synthesis in a packed bed reactor^14/15N]-L-citrulline.

In the examples of the present application, [ 2 ]^14/15N]The conversion of L-citrulline is calculated on the basis of carbon moles.