CN116004740A - Method for producing L-citrulline - Google Patents
Method for producing L-citrulline Download PDFInfo
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- CN116004740A CN116004740A CN202211727914.9A CN202211727914A CN116004740A CN 116004740 A CN116004740 A CN 116004740A CN 202211727914 A CN202211727914 A CN 202211727914A CN 116004740 A CN116004740 A CN 116004740A
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Abstract
The invention belongs to the technical field of amino acid production, and discloses a method for producing L-citrulline, which comprises the following steps: inoculating the prepared seed liquid into a fermentation medium according to the inoculation amount of 20%, fermenting and culturing for 50-55h, controlling the dissolved oxygen level at 20-25% to obtain L-arginine fermentation liquor, adding L-citrullinase prepared by a fermentation method, and reacting for 10-12h to obtain the L-citrulline fermentation liquor.
Description
Technical Field
The invention belongs to the field of L-citrulline production in the biological fermentation industry, and particularly relates to a method for producing L-citrulline.
Background
L-Citrulline (L-Citrulline) is a non-protein amino acid of formula C 6 H 13 N 3 O 3 L-citrulline is an important high-value fine chemical and widely applied to the industrial fields of foods, medicines, chemical industry, cosmetics and the like. Accompanied by pairing ofThe L-citrulline is studied continuously and deeply, so that the L-citrulline has wide application prospect in many aspects, has huge domestic and foreign demands and has wide market prospect.
The current production methods of L-citrulline mainly comprise a plant extraction method, an enzyme conversion method, a fermentation method and a chemical synthesis method. The plant extraction method has the advantages that the citrulline content in the plant is low, and the citrulline is influenced by factors such as seasons, production places and the like, so that the cost for extracting the citrulline is high, and the large-scale development is not facilitated; the chemical synthesis method is that L-arginine is hydrolyzed under alkaline condition to obtain L-citrulline, and the product contains optical enantiomer D-citrulline, which affects the quality of the product, and a large amount of waste water is generated in the production process, thus polluting the environment; the fermentation method takes glucose as a raw material and utilizes microbial strains to ferment and produce L-citrulline, and has the characteristics of wide raw material sources, low production cost and high product purity, but the breeding of high-yield strains also needs further research; the invention adopts an enzyme conversion method, utilizes arginine deiminase in microbial thalli to catalyze the conversion of L-arginine to generate L-citrulline, and has simple production process, high product concentration and purity and fewer purification steps than other preparation methods.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for producing L-citrulline, which can effectively improve the product yield and the yield and reduce the production cost.
The invention is realized by the following technical scheme.
A method for producing L-citrulline, comprising the steps of: step 1) seed liquid preparation, step 2) fermentation production process, step 3) L-citrullinase preparation and step 4) L-citrulline preparation.
Specifically, the method comprises the following steps:
step 1) seed liquid preparation: inoculating the strain producing L-arginine into a seed tank containing an L-arginine seed culture medium, wherein the pH is 6.8+/-0.1, the tank pressure is 0.05Mpa at 28 ℃, and the stirring rotation speed is 400rpm; when DO is reduced to about 30%, the rotation speed is sequentially increased from 400rpm to 3m 3 H→500rpm; air flow 3.0m 3 Culture under the condition of/h 12h, obtaining L-arginine seed solution;
step 2) fermentation production process: inoculating the L-arginine seed solution obtained in step 1) into a fermentation tank containing L-arginine fermentation medium, wherein the inoculum size is about 18%, the culture temperature is 30deg.C, the tank pressure is 0.05Mpa, pH7.0, and the air flow rate is 1.5m 3 And/h, stirring at 300rpm, and fermenting and culturing for 50-55h to obtain L-arginine fermentation liquor;
step 3) L-citrullinase preparation: inoculating the strain producing the L-citrullinase into an L-citrullinase seed culture medium, and culturing at 36-37 ℃ for about 4-6 hours at the OD600 = 4-6 to obtain an L-citrullinase seed solution; inoculating the L-citrullinase seed solution into a fermentation tank with an inoculum size of 5%, a culture temperature of 37 ℃, a pH value of 7.0, a tank pressure of 0.05Mpa, and an air flow: 2.0m 3 /h; stirring at 300rpm, and culturing for 22h to obtain L-citrullinase liquid;
step 4) L-citrulline preparation: adding the L-citrulline enzyme liquid obtained in the step 3) into the L-arginine fermentation liquid obtained in the step 2), wherein the adding amount is 10-15%, and stirring and reacting for 10-12h to obtain the L-citrulline. Adding L-citrullinase into the L-arginine fermentation broth with the addition amount of 10% -12%, and stirring and reacting for 10-12h to obtain L-citrulline.
Preferably, the L-arginine seed culture medium is 15g/L glucose, 4g/L magnesium sulfate heptahydrate, 10g/L beet molasses, 3.5g/L phosphoric acid, 3g/L potassium hydroxide, 2g/L yeast extract powder, 3.5g/L ammonium sulfate, 8mg/L ferrous sulfate, 16mg/L manganese sulfate, 2mg/L, VB copper sulfate 1 2mg/L, VH mg/L, and 0.2mL/L of bufomide.
Preferably, the formula of the L-arginine fermentation medium is glucose 18g/L, magnesium sulfate heptahydrate 3g/L, beet molasses 10g/L, phosphoric acid 1g/L, potassium hydroxide 1g/L, ammonium sulfate 12g/L, ferrous sulfate 5mg/L, manganese sulfate 5mg/L, copper sulfate 0.5mg/L, VB 1 1.5mg/L、VH 4mg/L、VB 5 3mg/L, and 0.5mL/L of polyether defoamer.
Preferably, the L-citrullinase seed culture medium is 12g/L of peptone, 18g/L of yeast extract powder, 5g/L of glycerol, 2.5g/L of monopotassium phosphate, 16.5g/L of dipotassium phosphate and 0.5mL/L of dichlord. The L-citrullinase fermentation medium comprises 0.8g/L of magnesium sulfate heptahydrate, 6g/L of yeast extract powder, 10g/L of peptone, 5g/L of dipotassium hydrogen phosphate, 2.5g/L of sodium chloride, 3g/L of ammonium sulfate, 2g/L of citric acid, 0.5g/L of ferric ammonium citrate, 10g/L of glycerol and 0.5mL/L of dichlord.
Preferably, said step 3) L-citrullinase preparation,
in the fermentation process, when dissolved oxygen and pH rise, the bottom sugar is exhausted, the material is fed about 5 hours, and the dissolved oxygen is controlled at 20-40%.
Preferably, said step 3) L-citrullinase preparation,
when OD600/100 reached 0.2, induction was started by addition of Inducer (IPTG) until fermentation was completed.
Preferably, the formula of the feed is as follows: 30g/L peptone, 75g/L yeast extract powder, 400g/L glycerol, and maintaining the pressure at 121 ℃ for 25min.
Preferably, the preparation method of the inducer comprises the following steps: firstly sterilizing water and a feed supplementing bottle, then weighing 100mg/L of Inducer (IPTG), and preserving the temperature for 5 minutes at 115 ℃.
The beneficial effects achieved by the invention include, but are not limited to, the following:
the L-citrulline product obtained by the technical method has high yield and purity, realizes simplified production steps, greatly improves production efficiency, reduces production cost, has simple production process, and is suitable for large-scale industrial production.
Detailed Description
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced with modification and alteration and combination of the products and methods described herein without departing from the spirit and scope of the invention. The present invention will be described in detail with reference to examples.
Example 1
A method of producing L-citrulline, comprising the steps of:
step 1) seed liquid preparation: the test strain used was Corynebacterium glutamicum ATCC 21493. Connecting one inclined plane of an eggplant bottle into a seed tank containing an L-arginine seed culture medium by using a flame protection method, wherein the pH value is 6.8+/-0.1, the tank pressure is 0.05Mpa at 28 ℃, and the stirring rotation speed is 400rpm; when DO is reduced to about 30%, the rotation speed is sequentially increased from 400rpm to 3m 3 H→500rpm; air flow 3.0m 3 Culturing for 12h under the condition of/h to obtain the L-arginine seed solution. The L-arginine seed culture medium comprises 15g/L glucose, 4g/L magnesium sulfate heptahydrate, 10g/L beet molasses, 3.5g/L phosphoric acid, 3g/L potassium hydroxide, 2g/L yeast extract powder, 3.5g/L ammonium sulfate, 8mg/L ferrous sulfate, 16mg/L manganese sulfate, 2mg/L, VB copper sulfate 1 2mg/L, VH mg/L, and 0.2mL/L of bufomide.
Step 2) fermentation production process: inoculating the seed solution obtained in step 1) into a fermentation tank containing L-arginine fermentation medium, wherein the inoculum size is about 18%, the culture temperature is 30deg.C, the tank pressure is 0.05Mpa, pH7.0, and the air flow is 1.5m 3 And/h, stirring at 300rpm, and fermenting and culturing for 50-55h to obtain the L-arginine fermentation liquor. The formula of the L-arginine fermentation medium comprises glucose 18g/L, magnesium sulfate heptahydrate 3g/L, beet molasses 10g/L, phosphoric acid 1g/L, potassium hydroxide 1g/L, ammonium sulfate 12g/L, ferrous sulfate 5mg/L, manganese sulfate 5mg/L, copper sulfate 0.5mg/L, VB 1 1.5mg/L、VH 4mg/L、VB 5 3mg/L, and 0.5mL/L of polyether defoamer. Through detection, the L-citrulline content is 108g/L.
Step 3) L-citrullinase preparation: pseudomonas putida ATCC4359 is selected as the test strain. Inoculating 1/2 branch of eggplant bottle inclined plane into sterilized seed culture medium, and culturing at 37deg.C with OD 600=4 for about 6 hr to obtain L-citrullinase seed solution; inoculating the L-citrullinase seed solution into a fermentation tank with an inoculum size of 5%, a culture temperature of 37 ℃, a pH value of 7.0, a tank pressure of 0.05Mpa, and an air flow: 2.0m 3 /h; stirring at 300rpm, and culturing for 22h to obtain L-citrullinase.
In the fermentation process, when dissolved oxygen and pH rise, the bottom sugar is exhausted, and the feeding is started about 5 hours, the feeding speed is adjusted according to the dissolved oxygen, and the dissolved oxygen is controlled to be 20-40%. The feed formula peptone 30g/L, yeast extract 75g/L, glycerol 400g/L, and pressure maintaining at 121 ℃ for 25min.
Preparation of inducer: firstly sterilizing water and a feed supplementing bottle, then weighing 100mg/L of Inducer (IPTG), and preserving the temperature for 5 minutes at 115 ℃. When OD600/100 reached 0.2, induction was started by addition of Inducer (IPTG) until fermentation was completed.
The L-citrulline enzyme seed culture medium is 12g/L of peptone, 18g/L of yeast extract powder, 5g/L of glycerol, 2.5g/L of monopotassium phosphate, 16.5g/L of dipotassium phosphate and 0.5mL/L of dichlormid. The L-citrullinase fermentation medium comprises 0.8g/L of magnesium sulfate heptahydrate, 6g/L of yeast extract powder, 10g/L of peptone, 5g/L of dipotassium hydrogen phosphate, 2.5g/L of sodium chloride, 3g/L of ammonium sulfate, 2g/L of citric acid, 0.5g/L of ferric ammonium citrate, 10g/L of glycerol and 0.5mL/L of dichlord.
Step 4) L-citrulline preparation: adding the L-citrullinase obtained in the step 3) into the L-arginine fermentation broth obtained in the step 2), wherein the adding amount is 10%, and stirring and reacting for 10-12h to obtain the L-citrulline. Under the above technological conditions, the L-arginine conversion rate reaches 100%.
Example 2
A method of producing L-citrulline, comprising the steps of:
step 1) seed liquid preparation: the test strain used was Corynebacterium glutamicum ATCC 21493. Connecting one inclined plane of an eggplant bottle into a seed tank containing an L-arginine seed culture medium by using a flame protection method, wherein the pH value is 6.8+/-0.1, the tank pressure is 0.05Mpa at 30 ℃, and the stirring rotation speed is 400rpm; when DO is reduced to about 30%, the rotation speed is sequentially increased from 400rpm to 3m 3 H→500rpm; air flow 3.0m 3 Culturing for 15h under the condition of/h to obtain the L-arginine seed solution. The L-arginine seed culture medium comprises 15g/L glucose, 4g/L magnesium sulfate heptahydrate, 10g/L beet molasses, 3.5g/L phosphoric acid, 3g/L potassium hydroxide, 2g/L yeast extract powder, 3.5g/L ammonium sulfate, 8mg/L ferrous sulfate, 16mg/L manganese sulfate, 2mg/L, VB copper sulfate 1 2mg/L, VH mg/L, and 0.2mL/L of bufomide.
Step 2) fermentation production process: inoculating the seed solution obtained in step 1) into a fermentation tank containing L-arginine fermentation medium, wherein the inoculum size is about 18% -20%, the culture temperature is 30deg.C, the tank pressure is 0.05Mpa, pH7.0, and the air flow is 1.5m 3 And/h, stirring speed is 300rpm,fermenting and culturing for 50-55h to obtain L-arginine fermentation liquor. The formula of the L-arginine fermentation medium comprises glucose 18g/L, magnesium sulfate heptahydrate 3g/L, beet molasses 10g/L, phosphoric acid 1g/L, potassium hydroxide 1g/L, ammonium sulfate 12g/L, ferrous sulfate 5mg/L, manganese sulfate 5mg/L, copper sulfate 0.5mg/L, VB 1 1.5mg/L、VH 4mg/L、VB 5 3mg/L, and 0.5mL/L of polyether defoamer. Through detection, the L-citrulline content is 115g/L.
Step 3) L-citrullinase preparation: pseudomonas putida ATCC4359 is selected as the test strain. Inoculating 1/2 branch of eggplant bottle inclined plane into sterilized seed culture medium, and culturing at 37deg.C with OD 600=6 for about 4 hr to obtain L-citrullinase seed solution; inoculating the L-citrullinase seed solution into a fermentation tank, wherein the inoculum size is 3%, the culture temperature is 37 ℃, the pH is 7.0, the tank pressure is 0.05Mpa, and the air flow is that: 2.0m 3 /h; stirring at 300rpm, and culturing for 24h to obtain L-citrullinase.
In the fermentation process, when dissolved oxygen and pH rise, the bottom sugar is exhausted, and the feeding is started about 5 hours, the feeding speed is adjusted according to the dissolved oxygen, and the dissolved oxygen is controlled to be 20-40%. The feed formula peptone 30g/L, yeast extract 75g/L, glycerol 400g/L, and pressure maintaining at 121 ℃ for 25min.
Preparation of inducer: firstly sterilizing water and a feed supplementing bottle, then weighing 100mg/L of Inducer (IPTG), and preserving the temperature for 5 minutes at 115 ℃. When OD600/100 reached 0.2, induction was started by addition of Inducer (IPTG) until fermentation was completed.
The L-citrulline enzyme seed culture medium is 12g/L of peptone, 18g/L of yeast extract powder, 5g/L of glycerol, 2.5g/L of monopotassium phosphate, 16.5g/L of dipotassium phosphate and 0.5mL/L of dichlormid. The L-citrullinase fermentation medium comprises 0.8g/L of magnesium sulfate heptahydrate, 6g/L of yeast extract powder, 10g/L of peptone, 5g/L of dipotassium hydrogen phosphate, 2.5g/L of sodium chloride, 3g/L of ammonium sulfate, 2g/L of citric acid, 0.5g/L of ferric ammonium citrate, 10g/L of glycerol and 0.5mL/L of dichlord.
Step 4) L-citrulline preparation: adding the L-citrullinase obtained in the step 3) into the L-arginine fermentation broth obtained in the step 2), adding 12 percent of the L-citrulline, and stirring and reacting for 10-12 hours to obtain the L-citrulline. Under the above technological conditions, the L-arginine conversion rate reaches 100%.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention.
Claims (9)
1. A method for producing L-citrulline, comprising the steps of: step 1) seed liquid preparation, step 2) fermentation production process, step 3) L-citrullinase preparation and step 4) L-citrulline preparation.
2. The method according to claim 1, characterized in that it comprises the steps of:
step 1) seed liquid preparation: inoculating the strain producing L-arginine into a seed tank containing an L-arginine seed culture medium, wherein the pH is 6.8+/-0.1, the tank pressure is 0.05Mpa at 28 ℃, and the stirring rotation speed is 400rpm; when DO is reduced to about 30%, the rotation speed is sequentially increased from 400rpm to 3m 3 H→500rpm; air flow 3.0m 3 Culturing for 12h under the condition of/h to obtain L-arginine seed solution;
step 2) fermentation production process: inoculating the L-arginine seed solution obtained in step 1) into a fermentation tank containing L-arginine fermentation medium, wherein the inoculum size is about 18%, the culture temperature is 30deg.C, the tank pressure is 0.05Mpa, pH7.0, and the air flow rate is 1.5m 3 And/h, stirring at 300rpm, and fermenting and culturing for 50-55h to obtain L-arginine fermentation liquor;
step 3) L-citrullinase preparation: inoculating the strain producing the L-citrullinase into an L-citrullinase seed culture medium, and culturing at 36-37 ℃ for about 4-6 hours at the OD600 = 4-6 to obtain an L-citrullinase seed solution; inoculating the L-citrullinase seed solution into a fermentation tank with an inoculum size of 5%, a culture temperature of 37 ℃, a pH value of 7.0, a tank pressure of 0.05Mpa, and an air flow: 2.0m 3 /h; stirring at 300rpm, and culturing for 22h to obtain L-citrullinase liquid;
step 4) L-citrulline preparation: adding the L-citrullinase liquid obtained in the step 3) into the L-arginine fermentation broth obtained in the step 2), wherein the adding amount is 10-15%, stirring and reacting for 10-12 hours to obtain L-citrulline, adding the L-citrullinase into the L-arginine fermentation broth, and stirring and reacting for 10-12 hours to obtain L-citrulline.
3. The method of claim 2, wherein the L-arginine seed medium is 15g/L glucose, 4g/L magnesium sulfate heptahydrate, 10g/L beet molasses, 3.5g/L phosphoric acid, 3g/L potassium hydroxide, 2g/L yeast extract, 3.5g/L ammonium sulfate, 8mg/L ferrous sulfate, 16mg/L manganese sulfate, 2mg/L, VB copper sulfate 1 2mg/L, VH mg/L, and 0.2mL/L of bufomide.
4. The method of claim 2, wherein the L-arginine fermentation medium is formulated as glucose 18g/L, magnesium sulfate heptahydrate 3g/L, beet molasses 10g/L, phosphoric acid 1g/L, potassium hydroxide 1g/L, ammonium sulfate 12g/L, ferrous sulfate 5mg/L, manganese sulfate 5mg/L, copper sulfate 0.5mg/L, VB 1 1.5mg/L、VH 4mg/L、VB 5 3mg/L, and 0.5mL/L of polyether defoamer.
5. The method according to claim 2, wherein the L-citrullinase seed medium is peptone 12g/L, yeast extract 18g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.5g/L, dipotassium hydrogen phosphate 16.5g/L, dichlord 0.5mL/L, and the L-citrullinase fermentation medium is magnesium sulfate heptahydrate 0.8g/L, yeast extract 6g/L, peptone 10g/L, dipotassium hydrogen phosphate 5g/L, sodium chloride 2.5g/L, ammonium sulfate 3g/L, citric acid 2g/L, ferric ammonium citrate 0.5g/L, glycerol 10g/L, and dichlord 0.5mL/L.
6. The method according to claim 2, wherein the step 3) L-citrullinase preparation,
in the fermentation process, when dissolved oxygen and pH rise, the bottom sugar is exhausted, the material is fed about 5 hours, and the dissolved oxygen is controlled at 20-40%.
7. The method according to claim 2, wherein the step 3) L-citrullinase preparation,
when OD600/100 reached 0.2, induction was started by addition of Inducer (IPTG) until fermentation was completed.
8. The method of claim 6, wherein the feed is formulated as follows: 30g/L peptone, 75g/L yeast extract powder, 400g/L glycerol, and maintaining the pressure at 121 ℃ for 25min.
9. The method of claim 6, wherein the inducer is prepared by the steps of: firstly sterilizing water and a feed supplementing bottle, then weighing 100mg/L of Inducer (IPTG), and preserving the temperature for 5 minutes at 115 ℃.
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