WO2021088604A1

WO2021088604A1 - Method for continuously preparing [14/15n]-l-citrulline by means of immobilized enzyme

Info

Publication number: WO2021088604A1
Application number: PCT/CN2020/120720
Authority: WO
Inventors: 黄钢; 李斌
Original assignee: 上海健康医学院
Priority date: 2019-11-06
Filing date: 2020-10-13
Publication date: 2021-05-14
Also published as: CN111518851A; CN111518851B

Abstract

Provided is a method for continuously preparing [^14/15N]-L-citrulline by means of an immobilized enzyme. The method comprises: suspending, in a packed bed reactor, a fusion protein cipA-arc formed by fixing an arginine deiminase arc to an inclusion body protein cipA; enabling a solution containing [^14/15N]-L-arginine to flow through the packed bed reactor at the flow rate of 0.3-0.5 BV/h and at the temperature of 20-55℃ for a reaction, and separating and purifying the reaction solution to obtain [^14/15N]-L-citrulline.

Description

Continuous preparation of an immobilized enzyme[ 14/15N]-L-citrulline method

Technical field

This application relates to a method for producing high-purity [ ^14/15 N]-L-citrulline, especially a method for the ^{production of [14/15} N]-L-arginine by using recombinant arginine deiminase The method of high purity [ ^14/15 N]-L-citrulline belongs to the technical field of enzymology and enzyme engineering.

Background technique

L-citrulline is a special amino acid. Participate in a variety of metabolic processes in the body, such as scavenging free radicals, indicators of foreign body rejection effects, vasodilation, stabilizing blood pressure, and diagnosing rheumatoid arthritis, anti-oxidation, etc., preventing prostate disease and improving sexual function, anti-aging and enhancing immunity, etc. , The application prospect is very broad.

The methods of producing L-citrulline include: chemical method, fermentation method, and enzymatic 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. The product contains the optical enantiomer D-citrulline, which affects the quality of the product and is produced during the production process. A large amount of waste water pollutes the environment; the difficulty of fermentation production 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; Under the action of arginine deiminase, L-arginine is converted into L-citrulline. This method has the advantages of strong specificity and high product concentration, but this method also has low catalyst utilization. That is, after each production, it must be re-fermented to prepare bacteria. This not only requires a large amount of raw materials, but also produces a large amount of waste water, which increases the cost of environmental protection treatment; and the impurities (a large amount of bacteria, a large number of bacteria, etc.) brought into the reaction system by the enzyme catalyst Impurities such as protein and various metal ions remaining in the fermentation broth), resulting in the separation and purification of the product in the L-citrulline post-treatment process, which has to go through a series of process steps such as bacteria removal, protein removal and separation column, which further increases Cost of production.

The chemical, fermentation, or enzymatic production of L-citrulline has many shortcomings, resulting in high production costs, which brings great difficulties to the actual promotion and application.

In order to overcome these problems, solutions for immobilized enzymes or cells have been proposed. The preparation methods of immobilized enzymes or cells are divided into two categories: physical methods and chemical methods. Physical methods include physical adsorption, embedding, etc. The advantage of physical immobilization of 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, because the embedded material or semipermeable membrane has a certain spatial or three-dimensional hindrance, it is not suitable for some reactions. Chemical methods include combining methods and cross-linking methods. The chemical method enzyme and the carrier are tightly bound, not easy to fall off, and have good stability, but the reaction conditions are intense, the operation is complicated, the control conditions are harsh, and the vitality loss is large.

In 2008, Zheng Pu (Zheng Pu, Ni Ye, Zhang Wen. Immobilized Pseudomonas cells in a packed bed reactor for continuous production of L-citrulline[J]. Journal of Food and Biotechnology, 2008, 27(5): 1673-1689) and others reported that the continuous production of L-citrulline by immobilized Pseudomonas cells in a packed bed reactor can be 0.0108g (hg) ^-1 (g citrulline produced per gram of cells per hour) Continuous 54-day operation under the conditions, but the fermentation production process of the bacteria is complicated, and the immobilized cell time is too long, the substrate concentration is low, and the yield is not high. Although the immobilized cells are simple, there are still cell fragments to release the contaminated proteins in the bacteria. The problems of other organic substances increase the difficulty of separation and purification of the products in the reaction system, and the immobilization of cells also increases the operation steps and increases the production cost.

Summary of the invention

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

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

(2) ^{Flow the solution containing [14/15} N]-L-arginine through the 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), the fusion protein containing the immobilized enzyme in step (1), is 9000-12000 U, preferably 10,000 U, and the step (2) contains [ ^14/15 N] -L- arginine solution ^[14/15 N] -L- arginine concentration 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.

Alternatively, the comprising ^[14/15 N] -L- arginine solution ^[14/15 N] -L- arginine concentration 1.0-2.5mol / L.

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

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

(1) Preparation of competent cells of Corynebacterium glutamicum;

(2) Using the recombinant plasmid pXMJ19-cipA-arc to transform the Corynebacterium glutamicum susceptible cells described in step (1) by electric shock to obtain the recombinant bacteria;

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

Optionally, the Corynebacterium glutamicum competent cell is prepared by the following method:

After culturing Corynebacterium glutamicum ATCC13032 in a solid medium containing LBG, pick a fresh strain and inoculate it in the LBG liquid medium. After culturing, transfer the activated bacterial solution to the LBG medium at an inoculum amount of 0.8-1.5% In the medium, continue to culture until the OD600 is 0.8-1.0; pre-cool the bacterial liquid with ice-water mixture, centrifuge, aspirate the supernatant and add glycerol, blow until the bacterial cells are suspended, centrifuge again, aspirate the supernatant, and add glycerol. Blow and suck until the cells are suspended, and then competent cells of Corynebacterium glutamicum can be obtained.

As a preferred solution, the Corynebacterium glutamicum ATCC13032 was streaked on a plate containing LBG solid medium and cultured in an incubator. After the bacteria grew, fresh strains were picked and inoculated into LBG liquid medium at a temperature of 20-40 Cultivate in a shaker with a rotation speed of 150-300r/min for 12-24 hours; transfer the activated bacterial solution to the LBG medium according to 1% of the inoculum, and in a shaker at a temperature of 20-40℃ with a rotation speed of 150-300r/min Cultivate to an OD600 of about 0.9; place the bacterial solution in an ice-water mixture pre-cooled for 15-20 minutes, and then distribute the pre-cooled bacterial solution into sterile centrifuge tubes in an ultra-clean table, centrifuge at 6000g at 4°C for 30 seconds, Place in water for 2 minutes; aspirate the supernatant in the centrifuge tube, add pre-cooled 10% glycerin to the centrifuge tube, and slowly blow with a pipette until the bacterial cells are suspended; centrifuge the suspension at 6000g at 4°C for 30 seconds, and centrifuge Aspirate the supernatant in the tube, add pre-chilled 10% glycerin to the centrifuge tube, and slowly blow with a pipette until the bacteria are suspended.

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

Optionally, the recombinant plasmid pXMJ19-cipA-arc is prepared by the following method for electric shock transformation of competent cells:

Take the competent cells of Corynebacterium glutamicum and the recombinant plasmid pXMJ19-cipA-arc and mix them evenly. After cooling on ice, under the same temperature conditions, the voltage is 1-5kV and the electric shock is 1-10ms; then add it at room temperature Transfer the LBG liquid medium to a centrifuge tube. After shaking culture, the obtained liquid is spread on a chloramphenicol resistant plate, a single colony is selected to extract the plasmid, and the insertion of the target fragment is confirmed by double enzyme digestion and PCR. Recombinant bacteria inoculation.

As a preferred solution, mix the competent cells and the recombinant plasmid pXMJ19-cipA-arc and cool on ice for 10 minutes; quickly add an ice-cold electric shock cup for electric shock, the electric shock condition is 2-4kV, time 3-7ms; take it out as soon as possible after the pulse is over In an electric shock cup, add LBG liquid medium at room temperature, transfer it to a centrifuge tube and gently shake it for 2 hours. Spread the resulting liquid on a plate containing 20 μg/mL chloramphenicol resistance; select a single colony to extract the plasmid, and then digest it with double enzymes or PCR confirms the insertion of the target fragment.

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

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

The recombinant bacteria were inoculated into LBG medium containing chloramphenicol, cultured on a shaker until the OD600 value of the bacteria reached 0.8-1.0, and isopropyl-β-D-thiogalactoside was added. After induction overnight, it was collected by centrifugation. Recombinant bacteria whole cells are washed with Tris-HCl buffer solution and resuspended in phosphate buffer solution, the cells are sonicated and centrifuged again, and the precipitate is the obtained cipA-arc fusion protein.

As a preferred solution, the successfully identified recombinant bacteria are inoculated into LBG medium with a final concentration of 20μg/mL chloramphenicol, the culture temperature is set to 20-40°C, the rotating speed of the shaker is 150-300r/min, and the bacteria are cultivated. When the OD600 value reaches 0.9, add IPTG with a final concentration of 1mM, and induce overnight at a temperature of 20-40℃ and a rotation speed of 150-300r/min; collect the recombinant cells by centrifugation at 4℃, wash the cells with buffer and resuspend in In another buffer, the cells were disrupted by ultrasound and centrifuged again at 4°C, and the precipitate was the obtained cipA-arc fusion protein.

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

Optionally, the buffer for washing is Tris-HCl; the other buffer for resuspension is phosphate buffer;

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

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

Optionally, the genetically engineered bacteria express arginine deiminase.

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

1) Introduce the cipA gene sequence into the HindIII site at the 5'end of the DNA, and introduce the SalI site at the 3'end to obtain a fragment with the gene sequence SEQ ID NO.1. After the synthesized fragment is sequenced, the target is digested with HindIII/SalI Gene and expression vector pXMJ19. After the digested product is recovered by gel, the target fragment and the vector are ligated, and the ligation product is transformed into E. coli DH5α competent cells to obtain the positive transformant pXMJ19-cipA;

2) Introduce the arc gene sequence into the XhoI site at the 5'end of the DNA and the SacI site at the 3'end to obtain a fragment with the gene sequence SEQ ID NO.2. After the synthesized fragment is sequenced, the target is digested with XhoI/SacI double enzyme Gene and expression vector pXMJ19-cipA. After the digestion product is recovered by gel, the target fragment and vector are ligated, and the ligation product is transformed into E. coli DH5α competent cells to obtain the positive transformant pXMJ19-cipA-arc, which contains spermine Genetically engineered bacteria for acid deiminase.

The deposited name of the genetically engineered bacteria containing arginine deiminase is Corynebacterium glutamicum SUMHS-2020.01, and the classification is named Corynebacterium glutamicum. This strain was deposited in Chaoyang District, Beijing, China on January 17, 2020 Beichen West Road, No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center, Chinese Microbial Culture Collection Management Committee, the collection 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 [ ^14/15 N]-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 [ ^14/15 N]-L-arginine further contains an ammonium acetate buffer solution, an ammonium formate buffer solution, an aqueous ammonium chloride solution or an aqueous ammonium bicarbonate 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 pH 4.5; the concentration of the ammonium bicarbonate aqueous solution is 0.3 mol/L and the pH is 8.5; the pH of the aqueous solution is 7.5.

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

Optionally, the separation and purification steps are as follows:

1) Collect the reaction liquid flowing out of the packed bed reactor, remove the buffer salt by nanofiltration, return to the reaction system, recycle, and collect the product concentrate;

2) Concentrate, crystallize, and dry the reaction liquid after step 1) under vacuum to obtain a white powdery solid, which is [ ^14/15 N]-L-citrulline with a purity of 99.5% or more.

In this application, "cipA gene sequence" refers to 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) The reported cipA gene sequence.

In this application, “arc gene sequence” refers to Kim et al. (Kim J E, Jeong D W, Lee H J. Expression, purification, and characterization of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 217962 in Escherichia J coli ]. Protein Expression and Purification, 2007, 53(1):0-15) reported the arginine deiminase (arc) gene sequence.

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

The beneficial effects that this application can produce include:

1) In this application, the gene of arginine deiminase was synthesized and cloned, and a high-yielding arginine deiminase engineering strain was constructed, and arginine deiminase was expressed in Corynebacterium glutamicum ；

2) In this application, arginine deiminase is immobilized on the inclusion body protein cipA to produce the inclusion body protein cipA-arc (ie cipA-arc fusion protein) with catalytic activity. The immobilization method is simple, fast, and low in cost. , High efficiency and easy to use;

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

4) The inclusion body protein cipA-arc provided in this application, that is, the arc-cipA immobilized fusion protein, can catalyze the conversion of [ ^14/15 N]-L-arginine to [ ^14/15 N]-L-citrulline by The fixed-bed reactor has simple post-processing, convenient product separation and purification, low cost, and easy to scale-up production, which ^{adds a new way for the enzymatic preparation of [14/15} N]-L-citrulline;

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

Detailed ways

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

Unless otherwise specified, the raw materials in the examples of this application are all purchased from the discovery platform, and the plasmid pXMJ19 is purchased from Wuhan Miaoling Biotechnology Co., Ltd.

Corynebacterium glutamicum ATCC13032 was purchased from Guangdong Province Microorganism Collection.

[ ^14/15 N]-L-citrulline determination method: HPLC determination of the product [ ^14/15 N]-L-citrulline, column C18, 5μm, 250mm×4.6mm; mobile phase is 5% methanol ; Flow rate 1mL/min; Detection wavelength 290nm; Column temperature is room temperature.

Definition of cipA-arginine deiminase activity: Under the conditions of 37℃ and pH 6.0, it can catalyze the conversion of [ ^14/15 N]-L-arginine to 1 μmol of [ ^14/15 N]-L every minute -The enzyme amount of citrulline is defined as one unit enzyme activity ((1U).

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

According to an embodiment of the present application, it mainly includes: 1) chemically synthesize the target gene (cipA, arc); 2) continuously connect the synthesized cipA and arc with the vector pXMG19 to construct the expression vector pXMJ19-cipA-arc; 3) PXMJ19-cipA-arc was introduced into Corynebacterium glutamicum ATCC13032 by electrotransformation; 4) Induced expression and separated the inclusion body protein cipA-arc (ie cipA-arc fusion protein); 5) Using the inclusion body protein cipA-arc in Continuously catalyze the synthesis of [ ^14/15 N]-L-citrulline from arginine in a packed bed reactor.

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

Example 1 Construction of genetically engineered bacteria containing arginine deiminase

1.1 According to 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 Company. The cipA gene sequence is as follows:

The synthesized gene sequence (SEQ ID NO.1) introduces HindIII site at the 5'end of the DNA and SalI site at the 3'end. After the synthesized fragment is sequenced, the target gene and the expression vector pXMJ19 (biological) are digested with HindIII/SalI. Wind). After the digested product is recovered by the gel, the target fragment and the vector are ligated, and the ligated product is transformed into E. coli DH5α competent cells, and the positive transformant is successfully identified and named pXMJ19-cipA.

1.2 According to the arginine deiminase (arc) gene sequence reported by Kim et al. (2007), 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 Company. The arc gene sequence is as follows:

The synthesized gene sequence (SEQ ID NO.2) introduces the XhoI site at the 5'end of the DNA and the SacI site at the 3'end of the DNA. After the synthesized fragment is sequenced, the target gene and the expression vector pXMJ19-cipA are digested with XhoI/SacI. After the digested product is recovered by the gel, the target fragment and the vector are ligated, and the ligated product is transformed into E. coli DH5α competent cells. After the positive transformant is successfully identified, it is named pXMJ19-cipA-arc, which is an arginine-containing dehydrogenase. Iminase genetically engineered bacteria.

Example 2 Expression of fusion protein cipA-arc

2.1 Preparation of Corynebacterium glutamicum competent cells

The Corynebacterium glutamicum ATCC13032 was streaked on a plate containing LBG solid medium and cultured in a 300C incubator. When the bacteria grew, fresh strains were picked and inoculated into LBG liquid medium. The temperature was 30°C and the rotation speed was 200r/ Incubate in a shaker for 12-24 hours. The activated bacteria solution was transferred to the LBG medium according to the 1% inoculum, and cultured in a shaker with a temperature of 30°C and a rotating speed of 200r/min until the OD600 was about 0.9. Pre-cool the bacteria liquid in a mixture of ice and water for 15-20 minutes, and then dispense the pre-cooled bacteria liquid into sterilized 50 mL centrifuge tubes in an ultra-clean table, centrifuge at 6000 g at 4°C for 30 seconds, and place in ice water for 2 minutes. Aspirate the supernatant in the centrifuge tube, quickly add 2.5 mL of pre-cooled 10% glycerin to the centrifuge tube, and slowly blow with a pipette until the bacterial cells are suspended. The suspension was centrifuged at 6000g for 30 seconds at 4℃, the supernatant in the centrifuge tube was aspirated, 500μL of pre-cooled 10% glycerol was quickly added to the centrifuge tube, and the cells were slowly sucked into suspension with a pipette, and this operation was repeated three times , You can get the competent cells of Corynebacterium glutamicum.

2.2 Recombinant plasmid pXMJ19-cipA-arc transforms competent cells by electric shock

Take 80 μL of competent cells and 2 μL of recombinant plasmid pXMJ19-cipA-arc and mix them and cool on ice for 10 minutes; quickly add an ice-cold electric shock cup for electric shock. The electric shock conditions are 2.5kV voltage and 5ms. Take out the shock cup as soon as possible after the pulse is over, add 1 mL of LBG liquid medium at room temperature, transfer to a centrifuge tube at 30°C, gently shake for 2 hours, and apply 200 μL to a plate containing 20 μg/mL chloramphenicol resistance. 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

Inoculate the successfully identified recombinant bacteria in the LBG medium containing the final concentration of 20μg/mL chloramphenicol, the culture temperature is set to 30℃, the shaker speed is 200r/min, and the final concentration is added when the bacteria OD600 reaches 0.9 It is 1 mM IPTG, induced overnight at 30°C and rotating speed 180r/min. Collect whole recombinant cells by centrifugation at 4°C, wash the cells twice with 50mM Tris-HCl, pH 7.0, resuspend in 50mM pH 6.5 phosphate buffer, sonicate the cells, and centrifuge again at 4°C to obtain the pellet. The inclusion body protein cipA-arc (ie cipA-arc fusion protein).

2.4 Determination of cipA-arc fusion protein activity by spectrophotometry

The specific color reaction of L-citrulline with diacetyl monooxime in a strong acid solution and the linear relationship between the absorbance of the reaction complex at 490 nm and the concentration of L-citrulline were used to determine the desublimation of cipA-arginine Enzymatic activity of aminase fusion protein. Prepare a substrate buffer solution ((pH 6.0, 50mM phosphate buffer) containing a final concentration of 200mM [ ^14/15 N]-L-arginine, take 2.8mL substrate solution, add 0.2mL enzyme solution, 37℃ React for 10 minutes. Dilute the enzyme reaction solution to an appropriate multiple (10-100 times), take 2 mL of the diluted reaction solution, and add 3 mL of mixed acid (volume ratio H ₂ SO ₄ : H ₃ PO ₄ =1: 3) solution, 0.5 The mixture of diacetyl-oxime and thiosemicarbazide, shake well, and immediately boil it in a water bath for 10 minutes, and measure its absorbance at 530nm. Definition of cipA-arginine deiminase fusion protease activity: at 37°C and pH 6.0 The amount of enzyme that catalyzes the conversion of [ ^14/15 N]-L-arginine to 1μmol citrulline per minute is defined as a unit enzyme activity ((1U), and the fusion protease activity is 10000U. Definition of specific enzyme activity: per mg protein The amount of enzyme activity contained in it (U/mg). The protein concentration was determined by 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 cultured to an OD600 of about 0.3, the remaining experimental conditions and experimental steps are the same as 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 reaches an OD600 of about 1.0, the remaining experimental conditions and experimental steps are the same as 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 reaches an OD600 of about 0.72, the other experimental conditions and experimental steps are the same as in Example 2.

The effect of different implementation conditions on the preparation of Corynebacterium glutamicum competent cells

实施例Example	培养温度(℃)Culture temperature (℃)	转速(r/min)Speed (r/min)	OD ₆₀₀ OD ₆₀₀	感受态细胞数量Number of competent cells
实施例2-2.1Example 2-2.1	3030	200200	0.90.9	正常normal
实施例3Example 3	2020	300300	0.30.3	弱weak
实施例4Example 4	3737	150150	1.01.0	正常normal
实施例5Example 5	4040	180180	0.720.72	弱weak

Example 6 Expression of fusion protein cipA-arc

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

Effects of Different Electric Shock Conditions on the Transformation Efficiency of Corynebacterium Glutamate Competent Cells

Example 8 Expression of fusion protein cipA-arc

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

Example 9 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.3 is 20°C, the speed of culture is 300r/min, the OD600 is about 0.5, the concentration of inducer is 0.5mM, the temperature of induction is 20°C, and the speed of induction is 200r/min. The rest of the experimental conditions and experimental steps are the same as in Example 2.

Example 10 Expression of fusion protein cipA-arc

Except that the culture temperature in step 2.3 is 25℃, the culture speed is 150r/min, the OD600 is about 1.0, the inducer concentration is 1.5mM, the induction temperature is 35℃, and the induction speed is 220r/min. The rest of the experimental conditions and experimental steps are the same as in Example 2.

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

Example 11 Transformation 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 packed column, and pack the immobilized fusion protein cipA-arc into the packed column.

Step 2: The immobilized fusion protein cipA-arc packed column catalyzes the synthesis of [ ^14/15 N]-L-citrulline:

The ^{concentration of [14/15} N]-L arginine is 2.5 mol/L ammonium acetate buffer solution (0.2 mol/l, pH 6.0) at a temperature of 30°C and a flow rate of 0.3 BV /h flows through the packed column. Under this reaction condition, ^{the conversion rate of [14/15} N]-L-arginine is 99.8%, and each liter of fermentation broth contains ^{435 g of [14/15} N]-L-citrulline, and the immobilized fusion protein cipA After 520h of arc transformation, the catalytic activity of the fusion protein cipA-arc is still stable, that is, the service life of the immobilized fusion protein cipA-arc is 520h.

Step 3. ^{The separation and purification of [14/15} N]-L-citrulline includes the following three steps:

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

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

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

Take a plexiglass column with a height-to-diameter ratio of 25 as a packed column, and pack the immobilized fusion protein cipA-arc into the packed column.

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

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

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

Example 13 Conversion of [ ^14/15 N]-L-Arginine and Production of [ ^14/15 N]-L-Citrulline

Take a plexiglass column with a height-to-diameter ratio of 30 as a packed column, and pack the immobilized fusion protein cipA-arc into the packed column.

The [ ^14/15 N]-L-arginine is a 1.5 mol/L ammonium chloride aqueous solution (0.3 mol/L, pH 4.5) at a temperature of 40°C and a flow rate of 0.5 BV/h flows through the packed column. Under this reaction condition, ^{the conversion rate of [14/15} N]-L-arginine is 99.5%, and each liter of fermentation broth contains ^{261 g of [14/15} N]-L-citrulline, and the immobilized fusion protein cipA After 530h of arc transformation, the catalytic activity of the fusion protein cipA-arc is still stable, that is, the service life of the immobilized fusion protein cipA-arc is 530h.

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

Example 14 Transformation of [ ^14/15 N]-L-Arginine and Production of [ ^14/15 N]-L-Citrulline

Take a plexiglass column with an aspect ratio of 40 as a packed column, and fill the packed column with the immobilized fusion protein cipA-arc.

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

Example 15 Transformation of [ ^14/15 N]-L-Arginine and Production of [ ^14/15 N]-L-Citrulline

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

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

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

The above are only a few embodiments of the application, and do not limit the application in any form. Although the application is disclosed as above with preferred embodiments, it is not intended to limit the application. Anyone familiar with the profession, Without departing from the scope of the technical solution of the present application, making some changes or modifications using the technical content disclosed above is equivalent to an equivalent implementation case and falls within the scope of the technical solution.

Claims

A method for continuous preparation of [ 14/15 N]-L-citrulline by immobilized enzyme, characterized in that the method comprises the following steps:

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

(2) The solution containing [ 14/15 N]-L-arginine is flowed through a packed bed reactor at a flow rate of 0.3-0.5 BV/h at 20-55 o C for reaction, and the reaction liquid is separated, After purification, [ 14/15 N]-L-citrulline can be obtained.
The method for continuous preparation of [14/15 N]-L-citrulline with an immobilized enzyme according to claim 1, wherein the fusion protein containing the immobilized enzyme in step (1) uses cipA as a carrier The catalytically active inclusion body protein cipA-arc obtained by immobilizing arginine deiminase arc on the inclusion body protein cipA, namely the cipA-arc fusion protein, the cipA-arc fusion protein is prepared by the following steps:

(1) Preparation of competent cells of Corynebacterium glutamicum;

(2) Using the recombinant plasmid pXMJ19-cipA-arc to transform the competent Corynebacterium glutamicum cells described in step (1) by electroporation to obtain recombinant whole cells;

(3) The recombinant bacteria obtained in step (2) are induced and expressed by genetically engineered bacteria to obtain recombinant bacteria whole cells, after ultrasonication and centrifugation, the resulting precipitate is the cipA-arc fusion protein.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 2, wherein the Corynebacterium glutamicum competent cell adopts the following preparation method:

After culturing Corynebacterium glutamicum ATCC13032 in a solid medium containing LBG, pick a fresh strain and inoculate it in the LBG liquid medium. After culturing, transfer the activated bacterial solution to the LBG medium at an inoculum amount of 0.8-1.5% In the medium, continue to culture until the OD600 is 0.8-1.0; pre-cool the bacterial liquid with ice-water mixture, centrifuge, aspirate the supernatant and add glycerol, blow until the bacterial cells are suspended, centrifuge again, aspirate the supernatant, and add glycerol. Blow and suck until the cells are suspended, and then competent cells of Corynebacterium glutamicum can be obtained.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 3, characterized in that the recombinant plasmid pXMJ19-cipA-arc is electro-shocked to transform competent cells using the following preparation method:

Take the competent cells of Corynebacterium glutamicum and the recombinant plasmid pXMJ19-cipA-arc and mix them evenly. After cooling on ice, under the same temperature conditions, the voltage is 1-5kV and the electric shock is 1-10ms; then add it at room temperature Transfer the LBG liquid medium to a centrifuge tube. After shaking culture, the obtained liquid is spread on a chloramphenicol resistant plate, a single colony is selected to extract the plasmid, and the insertion of the target fragment is confirmed by double enzyme digestion and PCR. Recombinant bacteria inoculation.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 4, wherein the method for inducing expression of the genetically engineered bacteria is as follows:

The recombinant bacteria were inoculated into LBG medium containing chloramphenicol, cultured on a shaker until the OD600 value of the bacteria reached 0.8-1.0, and isopropyl-β-D-thiogalactoside was added. After induction overnight, it was collected by centrifugation. Recombinant bacteria whole cells are washed with Tris-HCl buffer solution and resuspended in phosphate buffer solution, the cells are sonicated and centrifuged again, and the precipitate is the obtained cipA-arc fusion protein.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 2, wherein the recombinant plasmid pXMJ19-cipA-arc is prepared by the following method:

(1) Introduce the cipA gene sequence into the HindIII site at the 5'end of the DNA, and introduce the SalI site at the 3'end to obtain the fragment with the gene sequence SEQ ID NO.1. After sequencing, the synthesized fragment is digested with HindIII/SalI double enzyme Target gene and expression vector pXMJ19. After the digested product is recovered by gel, the target fragment and vector are ligated, and the ligation product is transformed into E. coli DH5α competent cells to obtain positive transformant expression vector pXMJ19-cipA;

(2) The arginine deiminase arc gene sequence is introduced into the XhoI site at the 5'end of the DNA and the SacI site at the 3'end of the DNA to obtain a fragment with the gene sequence SEQ ID NO.2. After the synthesized fragment is sequenced, The target gene and the expression vector pXMJ19-cipA were digested with XhoI/SacI. After the digested product was recovered by gel, the target fragment and the vector were ligated, and the ligation product was transformed into E. coli DH5α competent cells to obtain the positive transformant recombinant plasmid pXMJ19 -cipA-arc, which is a genetically engineered bacteria containing arginine deiminase.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 6, wherein the deposited name of the genetically engineered bacteria containing arginine deiminase is Gu Corynebacterium glutamicum SUMHS-2020.01, classified as Corynebacterium glutamicum, has been deposited on January 17, 2020 at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China. Chinese Microbial Strain Collection Management, Institute of Microbiology, Chinese Academy of Sciences The collection number of the General Microbiology Center of the Committee and the Culture Collection Center is CGMCC No.19404.
The method for continuous preparation of [14/15 N]-L-citrulline by immobilized enzyme according to claim 7, wherein the genetically engineered bacteria containing arginine deiminase expresses sperm Amino acid deiminase.
The method for continuous preparation of [14/15 N]-L-citrulline with an immobilized enzyme according to claim 1, wherein the activity of the fusion protease containing the immobilized enzyme in step (1) is 9000-12000 U said step (2) contains the [14/15 N] -L- arginine solution [14/15 N] -L- arginine concentration 1.0-2.5mol / L.
The method for continuous preparation of [14/15 N]-L-citrulline with an immobilized enzyme according to claim 1 , wherein the solution containing [14/15 N]-L-arginine further comprises Any one of ammonium acetate buffer solution, ammonium formate buffer solution, ammonium chloride aqueous solution, ammonium bicarbonate aqueous solution or pure aqueous solution;

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