Extraction and purification method for arginine-citrulline
阅读说明：本技术 一种精氨酸转瓜氨酸的提取纯化方法 (Extraction and purification method for arginine-citrulline ) 是由 岳明瑞 谢沛 曹华杰 郭永胜 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种精氨酸转瓜氨酸的提取纯化方法,包括以下步骤：(1)向含有精氨酸的底物液中加入精氨酸脱亚胺酶液进行转化,得到转化液；(2)将转化液进行陶瓷膜过滤,收集陶清液并稀释；(3)将稀释后的陶清液进行超滤,收集超清液；向超清液中加入活性炭进行脱色,得到第一脱色液；将第一脱色液进行电渗析,收集淡化液；(4)将淡化液浓缩得到第一浓缩液；将第一浓缩液离心,得到瓜氨酸粗品；(5)将瓜氨酸粗品重溶,得到重溶液；向重溶液中加入活性炭进行脱色,得到第二脱色液；(6)将第二脱色液浓缩得到第二浓缩液；将第二浓缩液搅拌结晶,离心,收集瓜氨酸晶体。采用本发明的分离纯化方法,可以显著提高瓜氨酸的收率和纯度。(The invention discloses an extraction and purification method of arginine-citrulline, which comprises the following steps: (1) adding an arginine deiminase solution into a substrate solution containing arginine for conversion to obtain a conversion solution; (2) filtering the conversion solution by a ceramic membrane, collecting and diluting a pottery clear solution; (3) carrying out ultrafiltration on the diluted pottery clear liquid, and collecting the ultra clear liquid; adding activated carbon into the super clear liquid for decoloring to obtain a first decoloring liquid; performing electrodialysis on the first decolorized solution, and collecting a desalted solution; (4) concentrating the desalted liquid to obtain a first concentrated liquid; centrifuging the first concentrated solution to obtain a citrulline crude product; (5) re-dissolving the crude citrulline product to obtain a heavy solution; adding activated carbon into the heavy solution for decoloring to obtain a second decolored solution; (6) concentrating the second destaining solution to obtain a second concentrated solution; and stirring the second concentrated solution for crystallization, centrifuging and collecting citrulline crystals. By adopting the separation and purification method, the yield and the purity of the citrulline can be obviously improved.)
1. The extraction and purification method of arginine-citrulline is characterized by comprising the following steps:
(1) adding arginine deiminase liquid into substrate liquid containing arginine, adjusting the pH value to 5.5-6.5, and converting for 3-5h to obtain conversion liquid;
(2) sterilizing the conversion solution, filtering by using a ceramic membrane, and collecting a ceramic clear solution; diluting the pottery clear liquid with pure water until the concentration of citrulline in the pottery clear liquid is less than or equal to 5 g/L;
(3) adding the diluted clear liquid of pottery into an ultrafiltration system for ultrafiltration, and collecting the ultra clear liquid; adding activated carbon into the super clear liquid for decoloring to obtain a first decoloring liquid; performing electrodialysis on the first decolorized solution, and collecting a desalted solution;
(4) concentrating the desalted liquid until the citrulline concentration is 400-450g/L to obtain a first concentrated liquid; centrifuging the first concentrated solution to obtain a citrulline crude product;
(5) re-dissolving the crude citrulline product by using pure water to obtain a heavy solution, wherein the concentration of citrulline in the heavy solution is 140-; adding activated carbon into the heavy solution for decoloring to obtain a second decolored solution;
(6) concentrating the second destaining solution to the citrulline concentration of 400-450g/L to obtain a second concentrated solution; and stirring the second concentrated solution for crystallization, centrifuging and collecting citrulline crystals.
2. The method according to claim 1, wherein in the step (1), the concentration of arginine in the substrate solution is 90-160 g/L; the volume ratio of the arginine deiminase liquid to the substrate liquid is 1: (8-12).
3. The method according to claim 1, wherein the pressure of the ultrafiltration treatment in the step (3) is 4kg, and the temperature of the feed liquid is controlled to be 40 ℃ or less.
4. The method as claimed in claim 1, wherein in the step (3), the activated carbon is added in an amount of 10% by weight of the super-clear liquid; decolorizing at 60 deg.C for 30 min.
5. The method as claimed in claim 1, wherein in the step (3), the pH of the first destaining solution is adjusted to 5.9-6.0, the electrodialysis is started, the temperature is controlled to be less than or equal to 40 ℃, and the pH is controlled to be 5.9-6.0; and when the conductance of the desalting chamber is less than or equal to 1000 mus/cm, closing the electrodialysis and discharging the desalted liquid.
6. The method according to claim 1, wherein in step (5), the redissolution temperature is 60 ℃.
7. The method as claimed in claim 1, wherein in the step (5), 5-10% o of activated carbon is added into the heavy solution, decolorization is carried out for 30min at 60 ℃, and light transmittance reaches more than 98%.
8. The method according to claim 1, wherein the method further comprises a step of washing the crude citrulline obtained in step (4) and the citrulline crystals obtained in step (6) with water; and (3) washing crude citrulline crude product with water to obtain crude mother liquor washing liquid and washing fine mother liquor washing liquid obtained by washing citrulline crystal with water to obtain the mother liquor washing liquid, and recycling the mother liquor washing liquid to the decoloring procedure before electrodialysis.
Technical Field
The invention relates to the technical field of bioengineering, in particular to an extraction and purification method for converting arginine into citrulline.
Background
L-citrulline is a special amino acid and is involved in various metabolic processes in vivo, such as the arginine-citrulline cycle. The research shows that: the L-citrulline can be widely applied to the aspects of medicines, foods, health products and the like. Can be used for treating diseases caused by L-arginine deficiency, preventing and treating prostate diseases, improving sexual function, resisting aging, and enhancing immunity.
The preparation method of the L-citrulline mainly comprises an extraction method, a fermentation method, a chemical method and an enzymatic method, wherein the extraction method and the fermentation method are used for respectively obtaining the citrulline from plants and microorganisms, but the problems of low concentration and the like exist; the control of the reaction process by a chemical method is difficult, and the problems of incapability of splitting DL-citrulline optical antipodes, low product purity, great environmental pollution and the like exist; the enzymatic synthesis of L-citrulline is mostly one-step enzymatic reaction, the one-step enzymatic synthesis of L-citrulline has the advantages of high substrate and product concentration, high conversion reaction specificity and no enantiomer structure, avoids the complex feedback regulation process in the fermentation production of L-citrulline, and realizes the one-step catalytic synthesis of high-concentration L-citrulline.
Arginine Deiminase (ADI) is called Arginine deiminase (EC3.5.3.6) for short, and is an Arginine degrading enzyme which catalyzes Arginine to generate L-citrulline and ammonia. Arginine deiminase from different species has different catalytic activity. ADI source strains currently applied to industrial production include pseudomonas proteus, enterococcus faecalis, pseudomonas putida and the like. However, the actual industrial application information for producing citrulline by one-step ADI enzymatic catalysis is less, mainly because ADI activity is low, most of the undeveloped sequences are expressed in the form of inclusion bodies in engineering strains, complex processes of inclusion body purification, denaturation and renaturation are needed for the development of the sequence, and the yield is low, thereby seriously restricting the actual industrial application.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a method for extracting and purifying citrulline from arginine.
In order to achieve the purpose, the invention adopts the following technical scheme:
an extraction and purification method of arginine-citrulline comprises the following steps:
(1) adding arginine deiminase liquid into substrate liquid containing arginine, adjusting the pH value to 5.5-6.5, and converting for 3-5h to obtain conversion liquid;
(2) sterilizing the conversion solution, filtering by using a ceramic membrane, and collecting a ceramic clear solution; diluting the pottery clear liquid with pure water until the concentration of citrulline in the pottery clear liquid is less than or equal to 5 g/L;
(3) adding the diluted clear liquid of pottery into an ultrafiltration system for ultrafiltration, and collecting the ultra clear liquid; adding activated carbon into the super clear liquid for decoloring to obtain a first decoloring liquid; performing electrodialysis on the first decolorized solution, and collecting a desalted solution;
(4) concentrating the desalted liquid until the citrulline concentration is 400-450g/L to obtain a first concentrated liquid; centrifuging the first concentrated solution to obtain a citrulline crude product;
(5) re-dissolving the crude citrulline product by using pure water to obtain a heavy solution, wherein the concentration of citrulline in the heavy solution is 140-; adding activated carbon into the heavy solution for decoloring to obtain a second decolored solution;
(6) concentrating the second destaining solution to the citrulline concentration of 400-450g/L to obtain a second concentrated solution; and stirring the second concentrated solution for crystallization, centrifuging and collecting citrulline crystals.
Preferably, in the step (1), the concentration of arginine in the substrate solution is 90-160 g/L; the volume ratio of the arginine deiminase liquid to the substrate liquid is 1: (8-12).
Preferably, in the step (1), the arginine deiminase solution is prepared by the following method:
inoculating the seed liquid of the arginine deiminase production strain into a fermentation culture medium for fermentation culture, wherein the temperature of the fermentation culture is 36-37 ℃, the pH value is 6.9-7.0, and the dissolved oxygen is 20-40%; performing fermentation culture until the OD of the fermentation liquid after being diluted by 100 times600When the value is 0.40-0.60, the temperature is reduced to 27-Adding IPTG into the system for induction culture at 29 ℃, and carrying out induction culture for 14-18 h;
monitoring the glycerol content of the system in the culture process, starting to add supplementary materials when the glycerol content of the system is less than or equal to 0.5g/L, and keeping the glycerol concentration in the system at 0.1-0.5g/L by feeding supplementary materials;
performing bacterium breaking treatment on the culture after induction culture, separating and collecting supernatant, and thus obtaining arginine deiminase liquid;
the fermentation medium comprises the following components: peptone 12g/L, glycerin 10g/L, yeast extract 8g/L, sodium chloride 3g/L, ammonium sulfate 2.5g/L, dipotassium phosphate trihydrate 4g/L, ferric ammonium citrate 0.3g/L, citric acid 2.1g/L, magnesium sulfate heptahydrate 0.5g/L, kanamycin sulfate 100 ppm;
the feed supplement comprises the following components: 400g/L of glycerol, 30g/L of peptone and 100g/L of yeast extract.
Preferably, in the step (3), the pressure of the ultrafiltration treatment is 4kg, and the temperature of the feed liquid is controlled to be less than or equal to 40 ℃.
Preferably, in the step (3), the adding amount of the activated carbon is 10 per mill of the weight of the super-clear liquid; decolorizing at 60 deg.C for 30 min.
Preferably, in the step (3), the pH of the first destaining solution is adjusted to 5.9-6.0, electrodialysis is started, the temperature is controlled to be less than or equal to 40 ℃, and the pH is 5.9-6.0; and when the conductance of the desalting chamber is less than or equal to 1000 mus/cm, closing the electrodialysis and discharging the desalted liquid.
Preferably, in step (5), the redissolution temperature is 60 ℃.
Preferably, in the step (5), 5-10 per mill of activated carbon is added into the heavy solution, decolorization is carried out for 30min at 60 ℃, and the light transmittance reaches more than 98%.
Preferably, the method further comprises the step of washing the citrulline crude product obtained in the step (4) and the citrulline crystal obtained in the step (6) with water; and (3) washing crude citrulline crude product with water to obtain crude mother liquor washing liquid and washing fine mother liquor washing liquid obtained by washing citrulline crystal with water to obtain the mother liquor washing liquid, and recycling the mother liquor washing liquid to the decoloring procedure before electrodialysis. The coarse mother liquor water washing liquid and the fine mother liquor water washing liquid after being washed by water are recycled to the decoloring procedure before electrodialysis, and the yield of citrulline is improved and the production cost is reduced by repeated application.
The invention has the beneficial effects that:
(1) the invention utilizes a recombinant bacillus subtilis system to produce arginine deiminase, the fermented product is subjected to bacteria breaking treatment, and the collected supernatant is used as arginine deiminase liquid. The arginine deiminase liquid used in the invention has high enzyme activity, and can obtain a substrate generation rate of more than 95% in a short time (3 h).
(2) For the conversion solution containing citrulline generated by catalysis of the arginine deiminase solution, the invention organically combines the technical means of ceramic membrane filtration, ultrafiltration, decoloration, electrodialysis and the like, and separates and purifies the citrulline; by adopting the separation and purification method, the yield and the purity of the citrulline can be obviously improved.
Drawings
FIG. 1: enzyme digestion verification result of the recombinant expression vector pWB980-Pg 3-arcA; in the figure, M is Marker, pWB980 in lane 1, pWB980-Pg3 in lane 2, arcA in lane 3 and pWB980-Pg3-arcA in lane 4.
FIG. 2: western bolt detection results of the arginine deiminase producing strain; in the figure, M is Marker, and lane 1 is a protein expressed by arcA of the target gene.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Description of terms:
the pure water used in the invention meets the requirements of the laboratory in GB 6682-2008 when other requirements are not marked.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples and comparative examples of the present invention are those conventional in the art and, unless otherwise specified, are commercially available.
Wherein: the bacillus subtilis is purchased from a Chinese microbial strain inquiry network (https:// www.biobw.org/China-strain/bio-81799.html), the number of the strain is bio-81799, and the strain name is bacillus subtilis 168.
Plasmid pWB980 was purchased from Beijing Tiannzze Gene technology, Inc.
Glycerol agar medium: 5g of peptone, 3g of yeast extract, 20g of glycerol and 15g of agar, and setting the volume to 1000 mL; HCl/NaOH adjusted to pH 7.0.
Primary seed culture medium: 5g of peptone, 3g of yeast extract and 20g of glycerol, wherein the volume is 1000 mL; adjusting the pH value to 7.0 by HCl/NaOH; kanamycin sulfate was added to give a final kanamycin sulfate concentration of 50 ppm.
Secondary seed culture medium: 5g of peptone, 3g of yeast extract and 20g of glycerol, wherein the volume is 1000 mL; adjusting the pH value to 7.0 by HCl/NaOH; kanamycin sulfate was added to give a final kanamycin sulfate concentration of 50 ppm.
Fermentation medium: peptone 12g/L, glycerin 10g/L, yeast extract 8g/L, sodium chloride 3g/L, ammonium sulfate 2.5g/L, dipotassium phosphate trihydrate 4g/L, ferric ammonium citrate 0.3g/L, citric acid 2.1g/L, magnesium sulfate heptahydrate 0.5g/L, kanamycin sulfate 100 ppm.
Example 1: preparation of arginine deiminase solution
(1) The plasmid pWB980 is subjected to double digestion treatment by EcoRI and KpnI, and a strong promoter sequence Pg3 (shown in SEQ ID NO. 1) is integrated on the plasmid pWB980 after double digestion treatment to obtain a plasmid pWB980-Pg3, wherein the nucleotide sequence of the plasmid pWB980-Pg3 is shown in SEQ ID NO. 2.
And carrying out enzyme digestion treatment on plasmid pWB980-Pg3 by using BamH I and SphI, and integrating the arcA gene (shown in SEQ ID NO. 3) optimized by codon on the plasmid pWB980-Pg3 after the enzyme digestion treatment to obtain a recombinant expression vector (pWB980-Pg 3-arcA).
The constructed recombinant expression vector is subjected to enzyme digestion verification by using two enzymes of BamHI and SphI, and the result is shown in FIG. 1. The results show that: the arcA gene (shown in SEQ ID NO. 3) has been successfully integrated into plasmid pWB980-Pg 3.
(2) The obtained recombinant expression vector (pWB980-Pg3-arcA) was introduced into Bacillus subtilis 168 to obtain a transformant. The transformant was spread on an LB plate containing 30. mu.g/ml kanamycin (kan), and a single colony that could grow was picked up as a positive transformant.
Positive transformants were inoculated into a glycerol agar medium containing 30. mu.g/ml kanamycin and cultured at 37 ℃ to OD600Slowly cooling to 28 deg.C, adding IPTG (to final concentration of 0.2mmol/L), and inducing culture for 16 h. After the induction culture is finished, the ultrasonic bacteria breaking, centrifugation and supernatant separation are carried out, and Western felt detection is adopted, and the result is shown in figure 2. An expression band is arranged at the position of 50.4KDa, and the molecular weight is consistent with the molecular weight obtained by theoretical calculation of the protein expressed by the target gene arcA inserted from an external source. This proves that: the arginine deiminase producing strain has been successfully constructed.
(3) Activating strains:
the constructed arginine deiminase-producing strain was streaked on a glycerol agar plate containing 30. mu.g/ml kanamycin, and cultured at 37 ℃ for 18 hours.
(4) Culturing the first-class strain:
inoculating 1-strain of the strain to the first-stage seed culture medium, culturing at 37 deg.C with Dissolved Oxygen (DO) of 30-40% and pH of 6.9-7.0 for 16 hr.
(5) And (3) secondary seed culture:
inoculating the first-stage seed solution into the second-stage seed culture medium at a concentration of 1% (volume fraction), culturing at 37 deg.C with Dissolved Oxygen (DO) of 30-40% until the OD is 100 times diluted600nmThe value is 0.2-0.4.
(6) Fermentation culture:
inoculating the secondary seed liquid of the arginine deiminase producing strain into a fermentation culture medium by 10 percent of inoculation amount (accounting for 10 percent of the weight of the fermentation culture medium) for fermentation culture, wherein the temperature of the fermentation culture is 37 ℃, the pH value is 6.9-7.0, and the Dissolved Oxygen (DO) is 20-40 percent; performing fermentation culture until the OD of the fermentation liquid after being diluted by 100 times600When the value is 0.5, cooling to 28 ℃, adding IPTG into the system to ensure that the final concentration of the IPTG in the system is 0.2mmol/L, and carrying out induction culture for 16h under the conditions of 28 ℃, pH6.9-7.0 and Dissolved Oxygen (DO) of 20-40%;
monitoring the glycerol content of the system in the culture process, and starting to add supplementary materials when the glycerol content of the system is less than or equal to 0.5g/L, wherein the supplementary materials comprise the following components: 400g/L of glycerol, 30g/L of peptone and 100g/L of yeast extract; the glycerol concentration in the system was maintained at 0.1-0.5g/L by fed-batch feeding.
(7) After the induction culture is finished, putting the tank, and performing bacterium breaking treatment on fermentation liquor after the tank is put by adopting a homogenizer, wherein the conditions of the homogenizing treatment are as follows: homogenizing pressure 12,000PSI, homogenizing flow rate 300L/Hr; homogenizing, centrifuging, and separating supernatant to obtain arginine deiminase solution.
Example 2: extraction and purification method for converting arginine into citrulline
The method comprises the following specific steps:
1. transformation of
Adding the arginine deiminase liquid prepared in the example 1 into a substrate liquid containing arginine, wherein the concentration of arginine in the substrate liquid is 150g/L, the volume ratio of the substrate liquid to the arginine deiminase liquid is 10:1, the pH value is adjusted to 6.0 during conversion, the temperature is 37 ℃, and the conversion is carried out for 3h, so as to obtain a conversion liquid.
2. Ceramic membrane filtration
2.1 ceramic membrane preheating: adding purified water into the ceramic membrane system, starting a ceramic membrane pump, and circularly preheating to 60-65 ℃.
2.2, material passing: sterilizing the conversion solution at 65 deg.C, filtering with ceramic membrane with aperture of 100nm and pressure of 0.5MPa, and collecting the pottery clear solution (filtrate). And adding pure water into the pottery clear liquid for dilution until the concentration of citrulline in the pottery clear liquid is less than or equal to 5 g/L.
2.3, cleaning: after the feed liquid was discharged, the system was washed with pure water and then with alkaline water of pH12, and after completion of the alkaline washing, the system was washed with pure water to a pH of about 7.0.
3. Ultrafiltration
3.1 material passing: and adding the diluted pottery clear liquid into an ultrafiltration system, performing ultrafiltration, and collecting the ultra clear liquid. The pressure is controlled to be 4kg, and the temperature of the feed liquid is not more than 40 ℃. Discharging the ultra-retained liquid in the system after the ultra-filtration is finished, cleaning the ultra-filtration system by using pure water, and collecting the ultra-retained liquid and the ultra-water washing liquid for the next batch.
3.2, cleaning: when the ultrafiltration system is cleaned, pure water is firstly used for water washing, then alkaline washing is carried out for 30min by alkaline water with the pH value of 12, and after the alkaline washing is finished, the system is cleaned to the pH value of about 7.0 by the pure water.
4. Primary decolorization
Adding 10 ‰ of activated carbon (the amount of activated carbon is 10 ‰ of the weight of the supernatantary liquid), decolorizing at 60 deg.C for 30min, and performing electrodialysis on the decolorized solution.
5. Electrodialysis
Pumping the decolorized solution into electrodialysis equipment, adjusting the pH of the decolorized solution to 5.92 with dilute sulfuric acid or ammonia water, starting the electrodialysis, controlling the pH to 5.92 and the temperature to be below 40 ℃; and when the conductance of the desalting chamber is less than or equal to 1000 mus/cm, closing the electrodialysis and discharging the desalted water.
6. Concentrating at one time
And starting a vacuum pump, pumping the desalted water into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450g/L to obtain a first concentrated solution.
7. Separation of
Centrifuging the first concentrated solution (4000r, 20min) to obtain crude citrulline, and washing the crude citrulline with pure water with the volume of 0.5-1 time that of the wet product; and (5) recycling the crude mother liquor washing liquid to a decoloring procedure before electrodialysis.
8. Redissolving
Re-dissolving the crude citrulline product by using pure water according to the concentration of 150g/L at the re-dissolving temperature of 60 ℃.
9. Secondary decolorization
Adding 5-10% of activated carbon into the heavy solution, decolorizing at 60 deg.C for 30min, and transmitting light to above 98% to obtain decolorized solution; fine filtering the decolorized solution, and concentrating.
10. Second concentration
And starting a vacuum pump, pumping the decolored liquid after fine filtration into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of the citrulline is 400-450g/L to obtain a second concentrated solution.
11. Separation of crystals
Slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, and centrifuging to obtain a refined citrulline product. Washing the crystal with pure water of 1-2 times of the volume of the wet product, detecting that no sulfate radical remains after washing to an outlet, and recycling the washing liquid of the seminal mother liquor to the decoloring procedure before electrodialysis.
12. Drying and packaging
Placing the citrulline fine product in an oven for drying at 60 ℃. And after the fine product is dried, crushing and packaging to obtain a finished product.
Comparative example 1:
1. transformation of
Adding the arginine deiminase liquid prepared in the example 1 into a substrate liquid containing arginine, wherein the concentration of arginine in the substrate liquid is 150g/L, the volume ratio of the substrate liquid to the arginine deiminase liquid is 10:1, the pH value is adjusted to 6.0 during conversion, the temperature is 37 ℃, and the conversion is carried out for 3h, so as to obtain a conversion liquid.
2. Ceramic membrane filtration
2.1 ceramic membrane preheating: adding purified water into the ceramic membrane system, starting a ceramic membrane pump, and circularly preheating to 60-65 ℃.
2.2, material passing: sterilizing the conversion solution at 65 deg.C, filtering with ceramic membrane with aperture of 100nm and pressure of 0.5MPa, and collecting the pottery clear solution (filtrate). And adding pure water into the pottery clear liquid for dilution until the concentration of citrulline in the pottery clear liquid is less than or equal to 5 g/L.
2.3, cleaning: after the feed liquid was discharged, the system was washed with pure water and then with alkaline water of pH12, and after completion of the alkaline washing, the system was washed with pure water to a pH of about 7.0.
3. Concentrating at one time
And starting a vacuum pump, pumping the pottery clear liquid into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450g/L to obtain a first concentrated solution.
4. Separation of
Centrifuging the first concentrated solution to obtain a crude citrulline product, and washing the crude citrulline product by using pure water with the volume of 0.5-1 time that of a wet product.
5. Redissolving
Re-dissolving the crude citrulline product by using pure water according to the concentration of 150g/L at the re-dissolving temperature of 60 ℃.
6. Decolorization of
Adding 5-10% of activated carbon into the heavy solution, and decolorizing at 60 deg.C for 30min to obtain decolorized solution; fine filtering the decolorized solution, and concentrating.
7. Second concentration
And starting a vacuum pump, pumping the decolored liquid after fine filtration into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of the citrulline is 400-450g/L to obtain a second concentrated solution.
8. Separation of crystals
Slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, and centrifuging to obtain a refined citrulline product. Washing the crystal with pure water of 1-2 times of the volume of the wet product, and detecting no sulfate radical residue after washing to an outlet.
9. Drying and packaging
Placing the citrulline fine product in an oven for drying at 60 ℃. And after the fine product is dried, crushing and packaging to obtain a finished product.
Comparative example 2:
1. transformation of
Adding the arginine deiminase liquid prepared in the example 1 into a substrate liquid containing arginine, wherein the concentration of arginine in the substrate liquid is 150g/L, the volume ratio of the substrate liquid to the arginine deiminase liquid is 10:1, the pH value is adjusted to 6.0 during conversion, the temperature is 37 ℃, and the conversion is carried out for 3 h.
2. Ultrafiltration
2.1 material passing: adding the conversion solution into an ultrafiltration system, carrying out ultrafiltration, and collecting the ultraclear solution. The pressure is controlled to be 4kg, and the temperature of the feed liquid is not more than 40 ℃. Discharging the ultra-retained liquid in the system after the ultra-filtration is finished, cleaning the ultra-filtration system by using pure water, and collecting the ultra-retained liquid and the ultra-water washing liquid for the next batch.
2.2, cleaning: when the ultrafiltration system is cleaned, pure water is firstly used for water washing, then alkaline washing is carried out for 30min by alkaline water with the pH value of 12, and after the alkaline washing is finished, the system is cleaned to the pH value of about 7.0 by the pure water.
3. Concentrating at one time
And starting a vacuum pump, pumping the pottery clear liquid into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450g/L to obtain a first concentrated solution.
4. Separation of
Centrifuging the first concentrated solution to obtain a crude citrulline product, and washing the crude citrulline product by using pure water with the volume of 0.5-1 time that of a wet product.
5. Redissolving
Re-dissolving the crude citrulline product by using pure water according to the concentration of 150g/L at the re-dissolving temperature of 60 ℃.
6. Decolorization of
Adding 5-10% of activated carbon into the heavy solution, and decolorizing at 60 deg.C for 30min to obtain decolorized solution; fine filtering the decolorized solution, and concentrating.
7. Second concentration
And starting a vacuum pump, pumping the decolored liquid after fine filtration into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of the citrulline is 400-450g/L to obtain a second concentrated solution.
8. Separation of crystals
Slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, and centrifuging to obtain a refined citrulline product. Washing the crystal with pure water of 1-2 times of the volume of the wet product, and detecting no sulfate radical residue after washing to an outlet.
9. Drying and packaging
Placing the citrulline fine product in an oven for drying at 60 ℃. And after the fine product is dried, crushing and packaging to obtain a finished product.
Comparative example 3:
1. transformation of
Adding the arginine deiminase liquid prepared in the example 1 into a substrate liquid containing arginine, wherein the concentration of arginine in the substrate liquid is 150g/L, the volume ratio of the substrate liquid to the arginine deiminase liquid is 10:1, the pH value is adjusted to 6.0 during conversion, the temperature is 37 ℃, and the conversion is carried out for 3h, so as to obtain a conversion liquid.
2. Primary decolorization
Adding 10 ‰ of active carbon (10 ‰ of the weight of the supernatantary liquid), and decolorizing at 60 deg.C for 30min to obtain decolorized solution.
3. Concentrating at one time
And starting a vacuum pump, pumping the pottery clear liquid into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450g/L to obtain a first concentrated solution.
4. Separation of
Centrifuging the first concentrated solution to obtain a crude citrulline product, and washing the crude citrulline product by using pure water with the volume of 0.5-1 time that of a wet product.
5. Redissolving
Re-dissolving the crude citrulline product by using pure water according to the concentration of 150g/L at the re-dissolving temperature of 60 ℃.
6. Secondary decolorization
Adding 5-10% of activated carbon into the heavy solution, and decolorizing at 60 deg.C for 30min to obtain decolorized solution; fine filtering the decolorized solution, and concentrating.
7. Second concentration
And starting a vacuum pump, pumping the decolored liquid after fine filtration into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of the citrulline is 400-450g/L to obtain a second concentrated solution.
8. Separation of crystals
Slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, and centrifuging to obtain a refined citrulline product. Washing the crystal with pure water of 1-2 times of the volume of the wet product, and detecting no sulfate radical residue after washing to an outlet.
9. Drying and packaging
Placing the citrulline fine product in an oven for drying at 60 ℃. And after the fine product is dried, crushing and packaging to obtain a finished product.
Comparative example 4:
1. transformation of
Adding the arginine deiminase liquid prepared in the example 1 into a substrate liquid containing arginine, wherein the concentration of arginine in the substrate liquid is 150g/L, the volume ratio of the substrate liquid to the arginine deiminase liquid is 10:1, the pH value is adjusted to 6.0 during conversion, the temperature is 37 ℃, and the conversion is carried out for 3h, so as to obtain a conversion liquid.
2. Electrodialysis
Pumping the conversion solution into electrodialysis equipment, adjusting the pH of the conversion solution to 5.92 by using dilute sulfuric acid or ammonia water, starting electrodialysis, controlling the pH to 5.92 and the temperature to be below 40 ℃; and when the conductance of the desalting chamber is less than or equal to 1000 mus/cm, closing the electrodialysis and discharging the desalted water.
3. Concentrating at one time
And starting a vacuum pump, pumping the pottery clear liquid into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450g/L to obtain a first concentrated solution.
4. Separation of
Centrifuging the first concentrated solution to obtain a crude citrulline product, and washing the crude citrulline product by using pure water with the volume of 0.5-1 time that of a wet product.
5. Redissolving
Re-dissolving the crude citrulline product by using pure water according to the concentration of 150g/L at the re-dissolving temperature of 60 ℃.
6. Decolorization of
Adding 5-10% of activated carbon into the heavy solution, and decolorizing at 60 deg.C for 30min to obtain decolorized solution; fine filtering the decolorized solution, and concentrating.
7. Second concentration
And starting a vacuum pump, pumping the decolored liquid after fine filtration into a rotary evaporation bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 75 ℃, starting cooling water for condensation, and concentrating until the concentration of the citrulline is 400-450g/L to obtain a second concentrated solution.
8. Separation of crystals
Slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, and centrifuging to obtain a refined citrulline product. Washing the crystal with pure water of 1-2 times of the volume of the wet product, and detecting no sulfate radical residue after washing to an outlet.
9. Drying and packaging
Placing the citrulline fine product in an oven for drying at 60 ℃. And after the fine product is dried, crushing and packaging to obtain a finished product.
The yield and purity of the finished product obtained in example 1 and comparative examples 1 to 4 were measured. Wherein, the yield is that the concentration of citrulline in the transformation liquid is directly measured firstly, and the amount of citrulline to be obtained is calculated; and then purifying to obtain a refined citrulline product, and calculating the yield according to the following formula:
yield (amount of citrulline fine product/amount of citrulline to be obtained) 100%.
The purity is the percentage of citrulline in the product.
The citrulline concentration in the conversion solution was measured in "spectrophotometric measurement of L-citrulline in the enzymatic conversion solution" (analytical laboratory, vol.24, 12 th, month 12 2005).
The method for measuring the content of citrulline in the finished product comprises the following steps:
1, reagent preparation:
1.10.1 mol/L perchloric acid standard solution preparation: 8.5mL of perchloric acid was measured, and 500mL of glacial acetic acid and 20mL of acetic anhydride were injected under stirring, followed by addition of 470mL of glacial acetic acid and mixing.
1.2 calibration: accurately weighing 90mg (weighed to be 0.0002g) of potassium hydrogen phthalate dried to constant weight, placing the potassium hydrogen phthalate in a triangular flask, adding 50mL of glacial acetic acid to dissolve the potassium hydrogen phthalate, adding 10 drops of an indicator, titrating the solution with prepared perchloric acid until the solution is dark green, and calibrating and taking an average value twice.
C1＝m3/0.2042*V2
In the formula:
C1-calibrated HClO4Concentration, mol/L
m3The mass of potassium hydrogen phthalate, g
V2The amount of perchloric acid standard solution, mL
0.2042——KHC8H4O4Millimolar mass of
1.30.2% alpha-naphthol phenyl carbinol
1.46% mercuric acetate-acetic acid solution: 6g of mercuric acetate was dissolved in 100mL of glacial acetic acid.
2, operation:
weighing 400mg of a sample dried to constant weight, weighing to 0.0002g, adding 3mL of formic acid, dissolving in 30mL of glacial acetic acid, adding 10mL of mercuric acetate-acetic acid solution and 2 drops of methyl violet indicator, titrating with 0.1mol/L perchloric acid standard solution until the sample turns blue, and simultaneously carrying out a blank test.
3, a calculation method:
in the formula:
x-content, is%
C-concentration of perchloric acid Standard solution, mol/L
▽0Titration of the blank consumes mL of perchloric acid standard solution
▽1Titration of the sample consumes mL of perchloric acid standard solution
m-sample weight, g
4, attention: if the temperature difference between the titration sample and the perchloric acid calibration exceeds 10 ℃, recalibration is needed, and if the temperature difference does not exceed 10 ℃, the perchloric acid solution concentration can be corrected according to the following formula:
in the formula:
0.0011-coefficient of expansion of glacial acetic acid
C1Calibration of the concentration of perchloric acid
The results of the yield and purity of citrulline purified in example 1 and comparative examples 1-4 were determined as follows:
table 1:
group of
Yield of
Purity of
Example 1
98％
99.5％
Comparative example 1
32％
80％
Comparative example 2
24％
84％
Comparative example 3
40％
45％
Comparative example 4
38％
28％
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Kazai Biotech Co., Ltd, Xintai City, Shantou City, Kazai Biotech Co., Ltd
<120> extraction and purification method for converting arginine into citrulline
<130> 2021
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 365
<212> DNA
<213> Artificial sequence
<400> 1
attcagcact ctttccacta tccctacagt gttatggctt gaacaatcac gaaacaataa 60
ttggtacgta cgatctttca gccgactcaa acatcaaatc ttacaaatgt agtctttgaa 120
agtattacat atgtaagatt taaatgcaac cgttttttcg gaaggaaatg atgacctcgt 180
ttccaccgga attagcttgg taccagctat tgtaacataa tcggtacggg ggtgaaaaag 240
ctaacggaaa agggagcgga aaagaatgat gtaagcgtga aaaatttttt atcttatcac 300
ttgacattgg aagggagatt ctttataata agaatgtgga attgtgagcg gataacaatt 360
cggta 365
<210> 2
<211> 3838
<212> DNA
<213> Artificial sequence
<400> 2
agaacctaaa aagaacgaat ttgaactaac tcataaccga gaggtaaaaa aagaacgaag 60
tcgagatcag ggaatgagtt tataaaataa aaaaagcacc tgaaaaggtg tctttttttg 120
atggttttga acttgttctt tcttatcttg atacatatag aaataacgtc atttttattt 180
tagttgctga aaggtgcgtt gaagtgttgg tatgtatgtg ttttaaagta ttgaaaaccc 240
ttaaaattgg ttgcacagaa aaaccccatc tgttaaagtt ataagtgact aaacaaataa 300
ctaaatagat gggggtttct tttaatatta tgtgtcctaa tagtagcatt tattcagatg 360
aaaaatcaag ggttttagtg gacaagacaa aaagtggaaa agtgagacca tggagagaaa 420
agaaaatcgc taatgttgat tactttgaac ttctgcatat tcttgaattt aaaaaggctg 480
aaagagtaaa agattgtgct gaaatattag agtataaaca aaatcgtgaa acaggcgaaa 540
gaaagttgta tcgagtgtgg ttttgtaaat ccaggctttg tccaatgtgc aactggagga 600
gagcaatgaa acatggcatt cagtcacaaa aggttgttgc tgaagttatt aaacaaaagc 660
caacagttcg ttggttgttt ctcacattaa cagttaaaaa tgtttatgat ggcgaagaat 720
taaataagag tttgtcagat atggctcaag gatttcgccg aatgatgcaa tataaaaaaa 780
ttaataaaaa tcttgttggt tttatgcgtg caacggaagt gacaataaat aataaagata 840
attcttataa tcagcacatg catgtattgg tatgtgtgga accaacttat tttaagaata 900
cagaaaacta cgtgaatcaa aaacaatgga ttcaattttg gaaaaaggca atgaaattag 960
actatgatcc aaatgtaaaa gttcaaatga ttcgaccgaa aaataaatat aaatcggata 1020
tacaatcggc aattgacgaa actgcaaaat atcctgtaaa ggatacggat tttatgaccg 1080
atgatgaaga aaagaatttg aaacgtttgt ctgatttgga ggaaggttta caccgtaaaa 1140
ggttaatctc ctatggtggt ttgttaaaag aaatacataa aaaattaaac cttgatgaca 1200
cagaagaagg cgatttgatt catacagatg atgacgaaaa agccgatgaa gatggatttt 1260
ctattattgc aatgtggaat tgggaacgga aaaattattt tattaaagag tagttcaaca 1320
aacgggccag tttgttgaag attagatgct ataattgtta ttaaaaggat tgaaggatgc 1380
ttaggaagac gagttattaa tagctgaata agaacggtgc tctccaaata ttcttattta 1440
gaaaagcaaa tctaaaatta tctgaaaagg gaatgagaat agtgaatgga ccaataataa 1500
tgactagaga agaaagaatg aagattgttc atgaaattaa ggaacgaata ttggataaat 1560
atggggatga tgttaaggct attggtgttt atggctctct tggtcgtcag actgatgggc 1620
cctattcgga tattgagatg atgtgtgtca tgtcaacaga ggaagcagag ttcagccatg 1680
aatggacaac cggtgagtgg aaggtggaag tgaattttga tagcgaagag attctactag 1740
attatgcatc tcaggtggaa tcagattggc cgcttacaca tggtcaattt ttctctattt 1800
tgccgattta tgattcaggt ggatacttag agaaagtgta tcaaactgct aaatcggtag 1860
aagcccaaac gttccacgat gcgatttgtg cccttatcgt agaagagctg tttgaatatg 1920
caggcaaatg gcgtaatatt cgtgtgcaag gaccgacaac atttctacca tccttgactg 1980
tacaggtagc aatggcaggt gccatgttga ttggtctgca tcatcgcatc tgttatacga 2040
cgagcgcttc ggtcttaact gaagcagtta agcaatcaga tcttccttca ggttatgacc 2100
atctgtgcca gttcgtaatg tctggtcaac tttccgactc tgagaaactt ctggaatcgc 2160
tagagaattt ctggaatggg attcaggagt ggacagaacg acacggatat atagtggatg 2220
tgtcaaaacg cataccattt tgaacgatga cctctaataa ttgttaatca tgttggttac 2280
gtatttatta acttctccta gtattagtaa ttatcatggc tgtcatggcg cattaacgga 2340
ataaagggtg tgcttaaatc gggccatttt gcgtaataag aaaaaggatt aattatgagc 2400
gaattgaatt aataataagg taatagattt acattagaaa atgaaagggg attttatgcg 2460
tgagaatgtt acagtctatc ccggcattgc cagtcgggga tattaaaaag agtataggtt 2520
tttattgcga taaactaggt ttcactttgg ttcaccatga agatggattc gcagttctaa 2580
tgtgtaatga ggttcggatt catctatggg aggcaagtga tgaaggctgg cgctctcgta 2640
gtaatgattc accggtttgt acaggtgcgg agtcgtttat tgctggtact gctagttgcc 2700
gcattgaagt agagggaatt gatgaattat atcaacatat taagcctttg ggcattttgc 2760
accccaatac atcattaaaa gatcagtggt gggatgaacg agactttgca gtaattgatc 2820
ccgacaacaa tttgattagc ttttttcaac aaataaaaag ctaaaatcta ttattaatct 2880
gttcagcaat cgggcgcgat tgctgaataa aagatacgag agacctctct tgtatctttt 2940
ttattttgag tggttttgtc cgttacacta gaaaaccgaa agacaataaa aattttattc 3000
ttgctgagtc tggctttcgg taagctagac aaaacggaca aaataaaaat tggcaagggt 3060
ttaaaggtgg agattttttg agtgatcttc tcaaaaaata ctacctgtcc cttgctgatt 3120
tttaaacgag cacgagagca aaacccccct ttgctgaggt ggcagagggc aggttttttt 3180
gtttcttttt tctcgtaaaa aaaagaaagg tcttaaaggt tttatggttt tggtcggcac 3240
tgattcagca ctctttccac tatccctaca gtgttatggc ttgaacaatc acgaaacaat 3300
aattggtacg tacgatcttt cagccgactc aaacatcaaa tcttacaaat gtagtctttg 3360
aaagtattac atatgtaaga tttaaatgca accgtttttt cggaaggaaa tgatgacctc 3420
gtttccaccg gaattagctt ggtaccagct attgtaacat aatcggtacg ggggtgaaaa 3480
agctaacgga aaagggagcg gaaaagaatg atgtaagcgt gaaaaatttt ttatcttatc 3540
acttgacatt ggaagggaga ttctttataa taagaatgtg gaattgtgag cggataacaa 3600
ttcggtacca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca 3660
tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca 3720
ctgctggcag gaggcgcaac tcaagctttt gcctcgagct cggtacccgg ggatcctcta 3780
gagtcgacct gcaggcatgc aagctagctt cagcacaatt ccaagaaaga cacgattt 3838
<210> 3
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<212> DNA
<213> Artificial sequence
<400> 3
gatcctatgt ccgctgaaaa acagaagtac ggtgtccact ccgaagccgg caaactgcgc 60
aaagtgatgg tttgctcgcc gggcctggcg cacaagcgcc tgacgccgag caactgtgac 120
gagctgctgt tcgacgatgt gatctgggtc gaccaggcca agcgtgacca tttcgacttc 180
gtcaccaaaa tgcgcgagcg cggcgtggat gtgctggaaa tgcacaacct gctgacagac 240
atcgtgcagc aacccgaagc cctcaagtgg atccttgacc gcaagattac ctctgacacc 300
gtgggtgtcg gcctgaccaa cgaggtgcgt agctggctcg aagggctgga accacgccac 360
ctggcagagt tcctgattgg cggcgtggca ggccaggacc ttccagtcag cgaaggcgcc 420
gaagtgatca agatgtacaa caagtacctc ggccactcca gcttcattct gccaccactg 480
cctaacaccc agttcacccg cgacaccacc tgctggatct acggtggcgt gacgctcaac 540
ccgatgtact ggcctgcgcg acgccaggaa accctgctga ccacagccat ttacaagttc 600
cacaaggaat tcaccggtgc tgacttccag gtctggtatg gcgacccgga caaggatcac 660
ggcaacgcca ccctagaggg cggcgacgtg atgccggtcg gcaagggcat cgtgctgatc 720
ggcatgggtg agcgcacctc gcgccatgcc atcggccaac tcgctcagaa cctgttcgag 780
aaaggcgcgg ccgagaaaat catcgtcgcc gggctgccga agtcccgcgc ggcgatgcac 840
ctggacaccg tgttcagctt ctgcgaccgc gacctggtga cggtattccc ggaggtggtg 900
aaagagatca agccattcat catcacccca gactccagca agccctatgg catgaacatc 960
gcgccgcagg atgcctcgtt cctcgaggtg gtatccgagc agttgctggg caagaaagac 1020
aagctgcgcg tggtcgagac cggcggcaac agctttgccg ccgagcgcga acagtgggat 1080
gacggcaaca acgtggtggc catcgagccg ggcgtggtca tcggctatga ccgcaacacc 1140
tacaccaaca ccctgctgcg caaggccggc atcgaggtca tcaccatcag cgccggcgaa 1200
ctgggccggg gccggggcgg cggccactgc atgacctgcc cgattgtgcg tgacccgatc 1260
gactacgcat g 1271