CN105177075A - Method for preparation of L-citrulline with arginine as raw material - Google Patents
Method for preparation of L-citrulline with arginine as raw material Download PDFInfo
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Abstract
The invention relates to a method for preparation of L-citrulline with arginine as the raw material. The method specifically includes the steps of: (1) acquisition of a substrate: subjecting an arginine ceramic membrane filtrate to adsorption by 732 amino type resin, conducting elution with 2.0N ammonia water to obtain an arginine positive column purified liquid; and (2) transformation culture: washing a arginine deiminase-containing thallus with normal saline once, performing centrifugation, then collecting the thallus, and transferring the thallus into a transformation liquid, which contains a 0.45mol/L acetic acid buffer solution and 0.5g/L CTAB, adding a 10% L-arginine purified liquid, and conducting heat preservation at 37DEG C for 24h, thus obtaining an L-citrulline transformation liquid. The method provided by the invention adopts an arginine fermented purified liquid as the transformation raw material, overcomes the bottleneck in production of citrulline by arginase transformation method, and has the advantages of cheap and easily available raw materials, simple process, and high product purity.
Description
Technical field
The present invention relates to a kind of is the method that Cit prepared by raw material with arginine, belongs to biological technical field.
Background technology
Right He Tailang in 1914 etc. are separated obtain citrulline from watermelon middle first times of squeezing the juice, and are after this seed amino acids by recognizing it with Tian Guangde.Citrulline is present in the seed of cucurbitaceous plant with free state, and up to now, success is from squeeze the juice by watermelon for people, and in wild watermelon leaf, walnut kernel, is separated in walnut seedling and seed and obtains citrulline H1.Citrulline is a kind of nonprotein amino acid, and the research of external scientist in citrulline is more, especially at Japan, US and European.At home, be but also just in the understanding stage to the research of citrulline, it is few that production method is not reported its detection method and physiological function research accordingly yet, only has a small amount of bibliographical information.The content of citrulline in TLCS Snakegourd Root such as Yue Rui.Studying the mould mutant strain induction of coarse spore with when cultivating, find that citrulline has obvious growth promoting function to this mutant strain.The L citrulline that the research such as Zheng Chunfu finds to increase cell external source can significantly improve the NO output that activated macrophage induces, and then reduces the infection rate of activated macrophage, and the propagation of toxoplasma tachyzoite in T suppression cell.Zeng little Feng etc. find that cyclic citrullinated peptid has important effect in the diagnosis of rheumatoid arthritis.
In the physiological function of citrulline, external scientist has studied and has shown that citrulline has some very important pharmacological functions, and as free radical scavenging effect, in the health care of human body, tool has very important significance; Vasorelaxation action, this is expected to become toxication, pyemic good medicine in treatment.Japan one scientist has developed a kind of citrulline novel polypeptide, and this novel polypeptide researchdevelopment is become anti-AIDS drug.Citrulline is considered to a kind of very effective antioxidant simultaneously, and this function has been applied to the every field such as makeup, medicine, protective foods.Numerous researchs of Abroad in Recent Years show, citrulline has much important physiological function, and as scavenging free radicals, allosome repelling effect indicator, vasorelaxation action, stabilizing blood pressure and diagnostics classes rheumatic arthritis, anti-oxidant etc., application prospect is very wide.
Primarily of three kinds in the preparation method of citrulline, external main employing fermentation method and Production by Enzymes, the separation method that separation and purification majority use ion-exchange etc. are conventional.
(1) chemical method: refer to that hydrolyzed under basic conditions L-arginine obtains Cit, process control is more difficult, containing optically active enantiomorph D-citrulline in product, affect quality product, a large amount of waste water is produced in production process, contaminate environment, but chemical method is the unique method of domestic current Cit suitability for industrialized production.
(2) difficult point of fermentative Production is that unit volume Cit productive rate is low, and only up to 1.7g/L, the cost extracting Cit from fermented liquid is higher.The advantage of fermentation method is that cost is low, and output is high, and the purity of product is high, does not have the generation of toxic substance in the production process of product, provides conveniently, reduce cost to product post-treatment.At present, be Japan to the country of fermentative Production citrulline most study, just have the research of this respect in the thirties in last century, reach certain level to the sixties.
(3) enzyme process: refer to that L-arginine is converted into Cit under the effect of arginine deiminase, working condition is gentle, and do not produce toxic substance, in transformation system, impurity is less, and extraction process is simple, pollutes few.This method is strong with specificity, that transformation efficiency is high microbial bacteria somatocyte is catalyzer, and catalysis arginine deiminase base generates L citrulline.Utilize enzymatic clarification citrulline, mostly adopt a step enzymatic reaction, thus can avoid feedback regulation effect complicated in the complete synthesis approach of citrulline, make citrulline can run up to higher concentration.
1973, IchifoChibata etc. ' 261 report PsudomonasputidaATCC4359, PseudomonasfluorescenIFO3081, PseudomonsovalisiAM1002, LeuconostoccitrovorumATCC8081 etc. can produce arginine deiminase, with L-arginine or DL arginine for substrate, citrulline concentration can be produced and reach more than 80 classes.The advantage of enzymatic clarification citrulline is that production concentration is high, and purification step is few, and without D-type optically active enantiomorph in product, production technique is simple, cost is low.But its shortcoming is current enzyme process to be difficult to meet industrial requirement, reason is that the activity of the arginine deiminase relying on pluck to obtain is lower, and transformation efficiency is lower, and the price of arginine raw material is high simultaneously, and the Production by Enzymes that have impact on citrulline is amplified.Therefore, cheap arginine cost of material and the acquisition of arginine deiminase are the keys of Production by Enzymes success.
Summary of the invention
A strain is the object of the present invention is to provide to produce the bacterial strain MQO-153 of arginine deiminase, the present invention passes through streptococcus faecium (streptococcus faecium is purchased from Beijing Culture Collection) chemomorphosis repeatedly, ultraviolet mutagenesis, and obtaining through a large amount of bacterial screenings the active high bacterial strain MQO-153 of arginine deiminase that a strain produces, its deposit number is CGMCCNo.10726; Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is April 21 in 2015; Preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The biological nature of bacterial strain MQO-153 is: bacterial strain MQO-153 is inoculated on slant medium and carries out cultivation 24h, bacterium shape circle or oval, can extend along the direction of chain, diameter 0.5-1.0 μm, great majority become two or short catenation, and on rich medium, bacterium colony is large and smooth, diameter 1-2mm, Quan Yuan, non-pigment.The Classification And Nomenclature of bacterial strain MQO-153 is streptococcus faecium (Streptococcusfaecalis).
Utilize bacterial strain MQO-153 to prepare the method for arginine deiminase, be specially microbial fermentation processes, step is as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-153 and cultivate, culture temperature is 30 DEG C, incubation time is 24h, and now, bacterium colony is large and smooth, and non-pigment accumulates;
Slant medium: slant medium (g/L): extractum carnis 5, peptone 10, sodium-chlor 5, agar 20; PH is 7.0-7.3; Be preferably 7.2; The sterilising temp of slant medium 115 DEG C, sterilization time 15min.
(2) enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, be inoculated into by phage solution and seed culture medium is placed in shaking table carries out enlarged culturing, inoculum size is 10% (v/v), the temperature of enlarged culturing is 30 DEG C, and incubation time is 18h, and shaking table amplitude is 85mm, shaking table frequency is 0-18h, and shaking speed is 100r/min.
Seed culture medium (g/L): glucose 30, extractum carnis 5, peptone 10, sodium-chlor 15, potassium primary phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005; PH is regulated to be 7.0 with sodium hydroxide; 250mL triangular flask liquid amount is 20mL, 1000mL triangular flask liquid amount is 150mL; Seed culture medium sterilising temp is 115 DEG C, and sterilization time is 15min.
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, culture condition is: culture temperature 30 DEG C, incubation time 28 hours, shaking table amplitude 85mm, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-28h, shaking speed 100r/min;
Fermention medium (g/L): glucose 3, yeast extract paste 10, extractum carnis 5, peptone 10, corn steep liquor 5, yeast extract paste 1, sodium-chlor 5, potassium primary phosphate 3, magnesium sulfate 0.5, L-arginine 5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005; PH is regulated to be 7.2 with sodium hydroxide, sterilising temp 121 DEG C, sterilization time 20min.
(4) 30L fermentor cultivation: the fermented liquid after fermentation culture in step (3) is inoculated in the substratum of 30L fermentor tank, inoculum size is 10% (v/v), control tank pressure 0.05MPa, cultivate 24 hours under 30 DEG C of conditions, mixing speed 600-800rpm, air quantity 1:0.8m
3/ m
3.min (vvm), control dissolved oxygen 30-40%, the aseptic saturated ammonia water management pH6.7-7.0 of whole process, it is 1% that midway benefit sugar controls residual sugar, and putting tank residual sugar is 0%, obtains arginine deiminase after fermentation.
The present invention produces citrulline by microbial enzyme method, is specially L-arginine essence and prepares Cit through the conversion of arginine deiminase.The present invention adopts a step enzymatic reaction, avoids feedback regulation effect complicated in the complete synthesis approach of citrulline, makes citrulline can run up to higher concentration.Concrete steps are as follows:
(1) acquisition of substrate: by arginine ceramic membrane filter liquid through 732 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid;
(2) transform and cultivate: by the thalline containing arginine deiminase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises the acetate buffer solution of 0.45mol/L and the CTAB of 0.5g/L, adds the L-arginine refined solution of 10%, and 37 DEG C of insulation 24h, obtain Cit conversion fluid.
(3) separation and purification:
Ion-exchange: conversion fluid is limpid to effluent liquid with water backwash resin after ammonia type 732 resin absorption, with 1.0N ammoniacal liquor wash-out, elutriant obtains ornithine refined solution after stream D335-OH type resin removing anionic impurity, and yield reaches 95%;
Nanofiltration: adopt 600-800 molecular weight nanofiltration membrane ornithine refined solution to obtain citrulline nanofiltration clear liquid, after deionization moisture 3 cleaning concentrates of 3 times of concentrated solution volumes, discard concentrated solution, collect the nanofiltration dialyzate containing citrulline product, this step yield reaches 98%;
Adjust pH, decolouring: the pH to 4.5 regulating citrulline nanofiltration clear liquid with hydrochloric acid, decolour with gac, the consumption of gac is 0.5% (massfraction) of citrulline nanofiltration clear liquid, bleaching temperature is 50 DEG C, bleaching time is 30min, obtain destainer with 0.22 μm of membrane filtration, destainer transmittance >=99%, yield reaches 99%;
Reverse osmosis concentration: citrulline destainer is concentrated into original volume half through reverse osmosis membrane and obtains the pre-concentration liquid after citrulline deamination, yield reaches 99%;
Condensing crystal: by pre-concentration liquid in vacuum tightness >=0.095, is concentrated into the concentrated solution of degree of enrichment content >=80% under vaporization temperature 50 DEG C of conditions, put by concentrated solution to 5 DEG C of refrigerators insulation 2h and obtain citrulline crystallization;
The acquisition of citrulline hydrochloride: the citrulline of crystallization is obtained citrulline hydrochloride wet product for 3 times with absolute ethanol washing after suction filtration, and yield reaches 85%;
Dry: citrulline hydrochloride wet product to be put into 55 DEG C of vacuum drying ovens dry 6h under >=-0.098 vacuum tightness and obtain citrulline hydrochloride finished product.
The present invention compared with prior art has the following advantages:
(1) screen a strain MQO-153, solve enzymatic translation technics enzyme source and the high restriction of enzyme cost;
(2) adopt arginine fermentation refined solution as conversion feedstock, overcome arginase conversion method and produce the bottleneck of citrulline, to be cheaply easy to get, technique is simple, and product purity is high;
(3) adopt the advanced treatment process such as membrane sepn, nanofiltration decolouring, membrane concentration to save energy consumption, save the consumption of gac, decrease the pollution to environment, reduce production cost, improve quality product.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The bacterial strain MQO-153 of arginine deiminase is produced in embodiment 1 one strain, the present invention passes through streptococcus faecium (streptococcus faecium is purchased from Beijing Culture Collection) chemomorphosis repeatedly, ultraviolet mutagenesis, and obtaining through a large amount of bacterial screenings the active high bacterial strain MQO-153 of arginine deiminase that a strain produces, its deposit number is CGMCCNo.10726; Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is April 21 in 2015; Preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment 2 utilizes bacterial strain MQO-153 to prepare the method for arginine deiminase, and concrete steps are as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-153 and cultivate, culture temperature is 30 DEG C, incubation time is 24h, and now, bacterium colony is large and smooth, and non-pigment accumulates;
Slant medium: slant medium (g/L): extractum carnis 5, peptone 10, sodium-chlor 5, agar 20; PH is 7.0-7.3; Be preferably 7.2; The sterilising temp of slant medium 115 DEG C, sterilization time 15min.
(2) enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, be inoculated into by phage solution and seed culture medium is placed in shaking table carries out enlarged culturing, inoculum size is 10% (v/v), the temperature of enlarged culturing is 30 DEG C, and incubation time is 18h, and shaking table amplitude is 85mm, shaking table frequency is 0-18h, and shaking speed is 100r/min.
Seed culture medium (g/L): glucose 30, extractum carnis 5, peptone 10, sodium-chlor 15, potassium primary phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005; PH is regulated to be 7.0 with sodium hydroxide; 250mL triangular flask liquid amount is 20mL, 1000mL triangular flask liquid amount is 150mL; Seed culture medium sterilising temp is 115 DEG C, and sterilization time is 15min.
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, culture condition is: culture temperature 35 DEG C, incubation time 28 hours, shaking table amplitude 85mm, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-28h, shaking speed 100r/min;
Fermention medium (g/L): glucose 3, yeast extract paste 10, extractum carnis 5, peptone 10, corn steep liquor 5, yeast extract paste 1, sodium-chlor 5, potassium primary phosphate 3, magnesium sulfate 0.5, L-arginine 5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005; PH is regulated to be 7.2 with sodium hydroxide, sterilising temp 121 DEG C, sterilization time 20min.
(4) 30L fermentor cultivation: the fermented liquid after fermentation culture in step (3) is inoculated in the substratum of 30L fermentor tank, inoculum size is 10% (v/v), control tank pressure 0.05MPa, cultivate 24 hours under 35 DEG C of conditions, mixing speed 600-800rpm, air quantity 1:0.8m
3/ m
3min (vvm), controls dissolved oxygen 30-40%, the aseptic saturated ammonia water management pH6.7-7.0 of whole process, and it is 1% that midway benefit sugar controls residual sugar, and putting tank residual sugar is 0%, obtains arginine deiminase thalline after fermentation.
The method of Cit is prepared in the smart conversion through arginine deiminase of embodiment 3L-arginine, and concrete steps are as follows:
(1) acquisition of substrate: by arginine ceramic membrane filter liquid through 732 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid;
(2) transform and cultivate: by the thalline containing arginine deiminase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises the acetate buffer solution of 0.45mol/L and the CTAB of 0.5g/L, adds the L-arginine refined solution of 10%, and 37 DEG C of insulation 24h, obtain Cit conversion fluid.
The separation and purification of embodiment 4L-citrulline conversion fluid
(1) ion-exchange: conversion fluid is limpid to effluent liquid with water backwash resin after ammonia type 732 resin absorption, with 1.0N ammoniacal liquor wash-out, elutriant obtains ornithine refined solution after stream D335-OH type resin removing anionic impurity, and yield reaches 95%;
(2) nanofiltration: adopt 600-800 molecular weight nanofiltration membrane ornithine refined solution to obtain citrulline nanofiltration clear liquid, after deionization moisture 3 cleaning concentrates of 3 times of concentrated solution volumes, discard concentrated solution, collect the nanofiltration dialyzate containing citrulline product, this step yield reaches 98%;
(3) pH, decolouring is adjusted: the pH to 4.5 regulating citrulline nanofiltration clear liquid with hydrochloric acid, decolour with gac, the consumption of gac is 0.5% (massfraction) of citrulline nanofiltration clear liquid, bleaching temperature is 50 DEG C, bleaching time is 30min, obtain destainer with 0.22 μm of membrane filtration, destainer transmittance >=99%, yield reaches 99%;
(4) reverse osmosis concentration: citrulline destainer is concentrated into original volume half through reverse osmosis membrane and obtains the pre-concentration liquid after citrulline deamination, yield reaches 99%;
(5) condensing crystal: by pre-concentration liquid in vacuum tightness >=0.095, is concentrated into the concentrated solution of degree of enrichment content >=80% under vaporization temperature 50 DEG C of conditions, put by concentrated solution to 5 DEG C of refrigerators insulation 2h and obtain citrulline crystallization.
The determination of conversion condition in test example 1 the present invention
(1) the determination temperature of reaction of temperature of reaction is on the impact of Cit output, under differing temps within the scope of 25 DEG C-42 DEG C, the situation of the conversion reaction l0h of immobilized cell and free cell, more Homocitrulline productive rate is higher for temperature, 37 DEG C reach the highest, and more than 37 DEG C, then productive rate declines.
(2) the determination pH of pH is on the impact of Cit output, substrate solution (pH4.0,5.0,8.0) under preparation condition of different pH, 10h is reacted at 37 DEG C, investigate the impact of pH on enzyme (product amount) alive, when pH is 6.5, concentration of substrate is the highest, and namely enzyme is lived the highest relatively.
(3) the determination rotating speed of rotating speed is on the impact of Cit output, and when rotating speed is 120r/min, citrulline output is maximum.But the intensity of rotating speed on particle causes very large impact, therefore rotating speed is adopted to be l00r/min.
(4) the determination concentration of substrate in concentration of substrate and reaction times and reaction times are on the impact of Cit output, and along with the increase of L-arginine concentration, Cit concentration reaches the maximum required time and extends gradually.When L-arginine concentration is 10%, Cell of Anmrobe 20h citrulline concentration reaches maximum, time expand production concentration no longer increase and tend towards stability; When L-arginine concentration continues to increase, the output of product Cit no longer increases and tends towards stability, and now increases concentration of substrate nonsensical, so select substrate L-arginine concentration to be 10%.
The mensuration of test example 2 arginine molar yield
Thalline is inoculated in 50mL liquid nutrient medium and cultivates 24h in 30 DEG C.The centrifugal 30min of 4000r/min at cultured fermented liquid 4 DEG C, abandoning supernatant.Thalline is transferred to 10mL acetate buffer solution, adds 0.15g arginine, at 37 DEG C, transform 24h.The centrifugal 10min of 6000r/min, measures the Cit concentration in supernatant liquor, and calculates the arginic molar yield y of substrate, represent the vigor of arginase with this.It is 98.2% that the present invention records arginine molar yield.
y＝nA/nB
Wherein nA is the amount of substance (mol) of Cit in conversion fluid; NB is arginic amount of substance (mol) original in conversion fluid.
The inspection of the Cit hydrochloride obtained in test example 3 the present invention
Pilot product is through the third party inspection of Pony Testing International Group, and indices meets US Federal Communication Committee, Cit hydrochloride USP standard, and main performance index is shown in Table 1:
Table 1
Claims (10)
1. be the method that Cit prepared by raw material with arginine, it is characterized in that, said method comprising the steps of:
(1) acquisition of substrate: by arginine ceramic membrane filter liquid through 732 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid;
(2) transform and cultivate: by the thalline containing arginine deiminase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises the acetate buffer solution of 0.45mol/L and the CTAB of 0.5g/L, adds the L-arginine refined solution of 10%, and 37 DEG C of insulation 24h, obtain Cit conversion fluid.
2. method according to claim 1, is characterized in that, the preparation process of described arginine deiminase is as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-153 and cultivate, culture temperature is 30 DEG C, incubation time is 24h;
(2) enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, phage solution is inoculated on seed culture medium, seed culture medium is placed in shaking table and carries out enlarged culturing, inoculum size is 10% (v/v), the temperature of enlarged culturing is 30 DEG C, and incubation time is 18h, and shaking table amplitude is 85mm, shaking table frequency is 0-18h, and shaking speed is 100r/min;
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, culture condition is: culture temperature 30 DEG C, incubation time 28 hours, shaking table amplitude 85mm, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-28h, shaking speed 100r/min;
(4) 30L fermentor cultivation: be inoculated in the substratum of 30L fermentor tank by the fermented liquid after fermentation culture in step (3), inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, controlling tank pressure is by force 0.05MPa, cultivates 24 hours, mixing speed 600-800rpm, air quantity 1:0.8m under 30 DEG C of conditions
3/ m
3min (vvm), control dissolved oxygen amount is 30-40%, the aseptic saturated ammonia water management pH6.7-7.0 of whole process, and it is 1% that sugar control residual sugar is mended in midway, and putting tank residual sugar is 0%, obtains arginine deiminase thalline after fermentation.
3. method according to claim 2, is characterized in that, in described step (1), slant medium (g/L) is: extractum carnis 5, peptone 10, sodium-chlor 5, agar 20.
4. method according to claim 3, is characterized in that, in described step (1), the pH of slant medium is 7.0-7.3; Preferably, pH is 7.2.
5. the method according to claim 3 or 4, in described step (1), the sterilising temp of slant medium is 115 DEG C, and sterilization time is 15min.
6. method according to claim 2, is characterized in that, in described step (2), seed culture medium (g/L) is: glucose 30, extractum carnis 5, peptone 10, sodium-chlor 15, potassium primary phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005.
7. method according to claim 6, is characterized in that, in described step (2), seed culture medium sodium hydroxide regulates pH to be 7.0, and sterilising temp is 115 DEG C, and sterilization time is 15min.
8. method according to claim 2, is characterized in that, in described step (3), fermention medium (g/L) is: glucose 3, yeast extract paste 10, extractum carnis 5, peptone 10, corn steep liquor 5, yeast extract paste 1, sodium-chlor 5, potassium primary phosphate 3, magnesium sulfate 0.5, L-arginine 5, ferrous sulfate 0.05, manganous sulfate 0.05, VB
1hCl0.005.
9. method according to claim 8, is characterized in that, in described step (3), fermention medium sodium hydroxide regulates pH to be 7.2, sterilising temp 121 DEG C, sterilization time 20min.
10. method according to claim 2, is characterized in that, the deposit number of described bacterial strain MQO-153 is CGMCCNo.10726; Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is April 21 in 2015; Preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
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| CN112500317A (en) * | 2020-12-07 | 2021-03-16 | 江苏优普生物化学科技股份有限公司 | Citrulline refining process |
| CN112608254A (en) * | 2020-12-29 | 2021-04-06 | 南通紫琅生物医药科技有限公司 | Method for preparing L-citrulline |
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