CN104726478A - Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli - Google Patents
Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli Download PDFInfo
- Publication number
- CN104726478A CN104726478A CN201510101168.6A CN201510101168A CN104726478A CN 104726478 A CN104726478 A CN 104726478A CN 201510101168 A CN201510101168 A CN 201510101168A CN 104726478 A CN104726478 A CN 104726478A
- Authority
- CN
- China
- Prior art keywords
- cit
- obtains
- arginine
- arginine deiminase
- escherichia coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses recombinant Escherichia coli for producing arginine deiminase at high yield. The recombinant Escherichia coli is collected with the serial number of CCTCC (China Center for Type Culture Collection) NO:M2015048 in CCTCC; an arginine deiminase gene is from Bacillus cereus which is bred after being subjected to induced mutation of N<+> ion beams; and then, a mutant strain for producing arginine deiminase at high yield is obtained through screening, wherein the collection number of the mutant strain is CCTCC NO:M2015047. The invention also discloses application of the recombinant Escherichia coli to production of L-citrulline and a method for producing L-citrulline. By using the recombinant Escherichia coli disclosed by the invention, more than 99% of arginine can be converted into L-citrulline, and the purity of a product can be up to more than 99%.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of arginine deiminase gene, express the recombination bacillus coli of this gene, the invention still further relates to this recombination bacillus coli in the application of producing in Cit and a kind of method of producing Cit.
Background technology
Cit has protection liver, lax blood vessel, and the treatment of sexual dysfunction, improves mental several functions of Denging, more and more comes into one's own in recent years in the field such as food, medicine, has development prospect widely.The production method of current Cit mainly contains plant extraction method, chemical synthesis, fermentation method and enzyme transforming process.Wherein plant extraction method content is low and extraction cost is high, is not suitable for scale operation; The environmental pollution of chemistry organic synthesis method is large and may residual toxicity material, there is potential security risk; Fermentation method production concentration is lower.By contrast, it is strong that enzyme transforming process has specificity, the features such as production concentration is high, with short production cycle, and cost is low.
Arginine deiminase (ADI) is present in a variety of microorganism, such as, be all found in the cells such as bacterium, mycoplasma, unicell green alga, yeast.The ADI obtained in certain micro-organisms is amino acid degradation enzyme, can be transformed into citrulline and ammonia by catalysis arginine.Current Production by Enzymes Cit many employings streptococcus faecium, clostridium perfringens, wine pyococcus, Pseudomonas aeruginosa etc. produce arginine deiminase, then become Cit with this enzymatic conversion arginine.
Along with molecular biological development, using gene engineering bacterium replaces the biosynthesizing that wild-type strain carries out arginine deiminase, has with short production cycle, and accumulation volume is large, the features such as in system, dopant species is few, and reaction efficiency is high, specificity is strong.
CN 104212753A discloses structure of a kind of arginine deiminase genetic engineering bacterium and uses thereof, and its arginine deiminase gene source is in pseudomonas putida (Pseudomonas putida).The Recombinant organism of acquisition being proceeded to after fermentation culture arginine concentrations is in the acetate buffer of 10%, can obtain the Cit of 99.4g/L.There is following shortcoming in this invention: the enzymic activity of this Recombinant organism is lower, wet thallus consumption higher (wet thallus of 1% need be added) required when causing transforming arginine, transformation time longer (the arginine substrate transforming 10% needs 4h); The temperature resistant capability difference (invert point can only control about 37 DEG C) of enzyme; Need transform under acetate salt buffer liquid system, be unfavorable for later stage separation and purification.
Summary of the invention
First object of the present invention is for above-mentioned defect, provides a kind of new arginine deiminase gene, makes the recombination bacillus coli of this gene of expression have higher arginine deiminase activity.
Second object of the present invention is to provide a kind of recombination bacillus coli.
3rd object of the present invention is to provide described recombination bacillus coli and is producing the application in Cit.
4th object of the present invention is to provide a kind of production method of Cit.
Above-mentioned purpose is achieved through the following technical solutions:
First, with the bacillus cereus obtained from soil (Bacillus cereus) for starting strain, breeding is carried out by N+ ion beam mutagenesis, screening obtains the bacillus cereus mutant strain 93-11 of a strain High-yield arginine deiminase bacterial, this bacterial strain was preserved on 01 20th, 2015 the China typical culture collection center (CCTCC) being positioned at Wuhan City, Hubei Province Wuhan University, and deposit number is CCTCCNO:M 2015047.This bacterial strain has applied for patent separately.
The bacillus cereus mutant strain obtained has higher arginine deiminase base enzymic activity, and the conversion of Arginine of more than 98% can be Cit by it, and the conversion of Arginine of more than 80% can only be become Cit by bacterium producing multi enzyme preparation of the prior art.
This mutant strain genomic dna of extracting, and design is with the primer of restriction enzyme site, by pcr amplification arginine deiminase gene, goal gene is cloned into carrier, transformed host cell, obtains recombination bacillus coli (Escherichia coli) the DH5 α I2-3 expressing described arginine deiminase gene.This recombinant escherichia coli strain was deposited on 01 20th, 2015 the China typical culture collection center (CCTCC) being positioned at Wuhan City, Hubei Province Wuhan University, and deposit number is CCTCC NO:M2015048.
This recombination bacillus coli has stronger arginine deiminase activity, produces Cit with it, significantly can reduce the consumption of thalline, shortens the time transformed, and improves transformation efficiency.
Finally, the invention provides a kind of production method of Cit, it comprises the following steps:
1) will China typical culture collection center be deposited in, deposit number be the recombination bacillus coli DH5 α I2-3 of CCTCC NO:M2015048 after seed culture and fermentation culture, the centrifugal wet thallus obtained containing arginine deiminase;
2) by step 1) wet thallus that obtains joins in the arginine aqueous solution, and regulate mixing solutions pH to be 6-7, under 37-60 DEG C of condition, react 0.5-4 hour;
3) separation and purification Cit from reaction solution.
Preferably, step 2) described in mixing solutions arginic concentration be 10-300g/L, the concentration of wet thallus is 0.5-5g/L.Under this condition, arginic transformation efficiency is higher, and transformation time is shorter.
Preferably, the method for described separation and purification Cit comprises the following steps:
1) micro-filtration: the ceramic microfiltration membrane by reaction solution by aperture being 0.02-1 μm, obtains micro-filtration dialyzate;
2) ultrafiltration: be 10-100 dust by aperture by micro-filtration dialyzate, retains the ultra-filtration membrane that molecule is 2000-10000, obtains ultrafiltration dialysis liquid;
3) electrodialysis: by ultrafiltration dialysis liquid through electrodialysis separating inorganic salts;
4) reverse osmosis membrane concentrates: by electrodialysis liquid through reverse osmosis membrane pre-concentration;
5) concentrated, crystallization: be 20-30, then crystallisation by cooling by reverse osmosis transparent liquid vacuum concentration to degree Beaume, collected by centrifugation crystallization, obtains a Cit crude product and mother liquor;
6) mother liquor is separated: by ion exchange resin column on mother liquor, with 2-5M ammoniacal liquor wash-out, elutriant vacuum concentration to degree Beaume is 20-30, then crystallisation by cooling, collected by centrifugation crystallization, obtains secondary Cit crude product;
7) crude product refining: by once with secondary Cit crude product mixing after add 10-100 times of weight pure water dissolve, then add activated carbon decolorizing, filter, it is 20-30 that filtrate is concentrated into degree Beaume, crystallisation by cooling, collected by centrifugation crystallization, drying obtains Cit and refines sterling.
Further preferably, described ion exchange resin column is D001 cation exchange resin column.
The invention has the beneficial effects as follows:
1) recombination bacillus coli that the present invention obtains has higher arginine deiminase base enzymic activity, transforms arginine and produces Cit, can significantly reduce thalline consumption, shorten transformation time, improve transformation efficiency with it.Wet thallus and arginic weight ratio is minimum is only 1:60, and bacillus cereus mutant strain before restructuring and arginic weight ratio are 1:20-30, in CN 104212753A, wet thallus and arginic weight ratio are 1:10; Within minimum 0.5 hour, can transform, and the bacillus cereus mutant strain before restructuring needs 10-50h, CN 104212753A needs 4 hours; Transformation efficiency reaches more than 99%, and the bacillus cereus mutant strain transformation efficiency before restructuring is more than 98%.
2) do not need in conversion fluid to add buffering salt ion, be conducive to later stage separation and purification, and CN104212753A need transform under acetate salt buffer liquid system.
3) heat-resisting ability of recombination bacillus coli is stronger, can react, and CN104212753A can only carry out enzyme reaction at 37 DEG C under 37-60 DEG C of condition.
4) owing to being recombination bacillus coli, do not need in fermention medium to add arginine, thus reduce fermentation costs, and restructuring before bacillus cereus mutant strain fermentation culture time need to add arginine.
5) product recovery rate adopting polishing purification method of the present invention to obtain is higher, and yield reaches more than 90%, and product purity is higher, and content reaches more than 99%.
6) the present invention can reduce the production cost of Cit, shortens the production cycle, enhances productivity.
Accompanying drawing explanation
Fig. 1 is the process flow sheet of separation and purification Cit from reaction solution.
Embodiment
The acquisition of embodiment 1 bacillus cereus mutant strain
1. prepare hair style bacterial strain
The strain bacillus cereus obtained in soil is as going out hair style bacterial strain, and access is containing in the substratum of 100ml, and 30-37 DEG C, 100-200r/min, cultivate 5-30h, then, get bacterium liquid 1ml, join in 9ml sterilized water, mixing, weaker concn is 10 times, and Using such method continues dilution 10
-2, 10
-3with 10
-4doubly, bacterium liquid is for subsequent use.
Described substratum composition is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium-chlor, surplus is deionized water, and pH is 6.0-7.5.
2.N ion beam mutagenesis
Get bacterium liquid 0.5ml prepared by step 1 to be applied in sterile petri dish and to dry up, adopt TITAN ion beam implanter to carry out N ion beam mutation to thalline, energy is 30keV, and implantation dosage is 1-5 × 10
15ion/cm
2, use the thalline on 5ml sterile water wash culture dish after injection again, dilution 10
3get 50 μ l applying solid culture medium flat plates doubly, in temperature 30-37 DEG C of constant incubator, cultivate 5-30h.
The composition of described solid medium is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium-chlor, 0.1-30% agar, and surplus is deionized water, and pH is 6.0-7.5.
3. the screening of High-yield arginine deiminase bacterial mutant strain
The inoculation selected after 200-1000 strain mutagenesis from flat board cultivates 5-30h to slant medium.Select the bacterial classification on a ring slant medium, in access 50-200ml seed culture medium, 30-37 DEG C, 100-200r/min, cultivate 5-30h.Seed liquor is seeded in 100-300ml fermention medium according to the ratio of 1-10%, 30-37 DEG C, 100-200r/min, get fermented liquid centrifugal after cultivation, obtain the resting cell containing arginine deiminase, fermentation culture duration 5-16h, comparatively starting strain fermentation time reduction 20%.
Resting cell is joined in arginine solution, adjust PH5-7, transform at 30-37 DEG C, synthesis Cit.By the reaction solution after conversion respectively through deactivation, dilution, by Agilent liquid chromatograph, respectively arginine, Cit are carried out quantitatively.Select the mutant strain 93-11 that a strain Cit output is the highest, the resting cell of fermentation can make the conversion of Arginine of more than 98% be Cit, and starting strain can only make the conversion of Arginine of about 30% be Cit.
This bacterial strain was preserved in China typical culture collection center on 01 20th, 2015, and deposit number is CCTCC NO:M 2015047.
The composition of described slant medium is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium-chlor, 0.1-30% agar, and surplus is deionized water, and pH is 6.0-7.5.
Described seed culture medium composition is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium-chlor, 0.1-10% glycerine, surplus is deionized water, and PH is 6-7.
Described fermention medium composition is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium-chlor, 0.1-10% glycerine, 0.1-5% arginine, surplus is deionized water, and PH is 6-7.
Described liquid-phase chromatographic analysis condition is as follows: INSTRUMENT MODEL is for wearing peace U3000, chromatographic column: (250*4.6) mm 5um NH
2-X, column temperature: 30 DEG C, wavelength 205nm, moving phase: 0.02M potassium primary phosphate (PH5.6 includes 1% ammoniacal liquor): acetonitrile=40:60, flow velocity 1.3ml/min
The extraction of embodiment 2 genome, object fragment amplification and construction of genetic engineering
1. reagent and material: intestinal bacteria (Escherichia coli) DH5 α, BL21, pMD19-Tsimple vector, pET28a carrier is by this Laboratories Accession.
Experiment reagent: archaeal dna polymerase (KOD DNA Polymerase) is purchased from TOYOBO; Restriction enzyme (EcoRI and NotI), T4DNA Ligase, dNTPs, DNA marker DL2,000, plasmid Mini Kit, DNA purification kit is all purchased from Takara company.Bacterial genomes extracts test kit purchased from Tian Gen company, and penbritin, sulfuric acid card receive mycin, IPTG purchased from Biosharp company, and all the other chemical reagent are analytical pure.
2. the extraction of bacillus cereus genomic dna
Selecting deposit number is that the mono-colony inoculation of bacillus cereus 93-11 of CCTCC NO:M 2015047 is to incubated overnight in the LB liquid nutrient medium of 1-4ml; Get the incubated overnight bacterium liquid of 1-4ml, centrifugal 2 minutes of 10,000rpm, abandons supernatant; Wet thallus sky kan gene group extracts test kit extracting genomic dna.
3. arginine deiminase (ADI) gene PCR amplification
Primer according to the gene design band restriction enzyme site of Bacillus cereus ATCC 10987:
FP-arcA 5’-CGG
AATTCATGAAACATCCGATACATG-3’，
RP-arcA 5’-ATA
AGAATGCGGCCG CTAAATATCTTTAC-3’
Add EcoRI and NotI double enzyme site, with the DNA extracted for template, carry out pcr amplification with the KOD enzyme of high-fidelity and obtain arginine deiminase gene.
PCR reaction system (50 μ l): 5 × PCR buffer 10 μ l; DNTP 5 μ l; The each 1.5 μ l of primers F P-arcA and RP-arcA (10 μMs); Template 1 μ l; KOD enzyme 1 μ l; Add water to 50 μ l.
PCR reaction conditions: 95 DEG C of 5min; 95 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 2min, 30 circulations; 68 DEG C of 10min.1% agarose gel electrophoresis detects and purified pcr product.
4. the Construction and identification of target gene clone library
According to pMD19-T simple vector test kit operation instructions, linking objective gene and carrier T, Transformed E .coli DH5 α, coat the flat board containing penbritin, IPTG and X-gal, utilize blue hickie to screen positive transformant, and select part transformant to deliver to the order-checking of Wuhan Tian one Hui Yuan company.
5. target gene clone and qualification
In the sequencing result of clone library, select the transformant carrying goal gene, 37 DEG C of shake-flask culture that spend the night, extract recombinant plasmid, utilize EcoRI and NotI double digestion, reclaim goal gene through agarose gel electrophoresis purifying.Utilize EcoRI and NotI double digestion pET28a carrier simultaneously, and sepharose reclaims support products, utilize T4 ligase enzyme 16 DEG C of enzymes that spend the night to connect pET28a carrier and goal gene, product conversion E.coli BL21, select part transformant to deliver to the order-checking of Wuhan Tian one Hui Yuan company.Check order the correct genetic engineering bacterium be containing recombinant plasmid (pET-ADI), and sequencing result is as shown in SEQID NO:1.
By this genetic engineering bacterium called after DH5 α I2-3, and be preserved in China typical culture collection center on 01 20th, 2015, deposit number is CCTCC NO:M 2015048.
The fermentation culture of embodiment 3 recombinant bacterium
1. seed culture:
The engineering strain access 250ml eggplant type bottle LB solid slant culture 16-24h preserved by glycerine, then washes with sterilizing, gets about 25ml and access in seed culture medium.
Seed culture medium: corn steep liquor 2-10%, monosodium glutamate 1-10%, MgSO
40.01-0.1%, KH
2pO
40.01-0.1%, pH are 4.0-7.5, and deionized water is prepared;
Seed culture condition: bacterial strain in 30-37 DEG C, in seed culture medium, cultivate 5-30h under the hunting speed of 100-300rpm, obtain kind of a daughter bacteria;
2. fermentation culture
Fermention medium: corn steep liquor 3-15%, monosodium glutamate 2-12%, MgSO
40.01-0.1%, KH
2pO
40.01-0.1%, pH are 4.0-7.5, and deionized water is prepared;
Fermentation condition: inoculum size 1-10%, 30-37 DEG C, cultivate 0.5-4h in the fermentation medium under the condition of rotating speed 150-450rpm after, adding final concentration is after the lactose-induced 4-22h of 0.1-2mg/ml, and centrifugal fermented liquid obtains the wet thallus being rich in arginine deiminase.
Adopt above-mentioned cultural method, (1) thalline can be induced great expression arginine deiminase, and in the wet thallus of results, arginine deiminase activity is the highest; (2) thalline can obtain optimum nutrition, and grow vigorous, fermentation time is the shortest and harvest yield is the highest.
Embodiment 4 produces Cit
1. transform arginine with the wet thallus being rich in arginine deiminase
Joined by wet thallus in the arginine aqueous solution, make arginine concentration of substrate be 300g/L, the concentration of wet thallus is 5g/L, and regulator solution pH is 6, under temperature 60 C condition, react 4h.
After testing and calculate, the conversion of Arginine of 99.2% is Cit.
2. separation and purification Cit from reaction solution
1) micro-filtration: the ceramic microfiltration membrane by conversion fluid by aperture being 0.02 μm, working pressure 7kPa, the thalline in removing conversion fluid and high molecular weight protein material, obtain micro-filtration dialyzate;
2) ultrafiltration: micro-filtration dialyzate is 100 dusts by aperture, retains the PVDF ultrafiltration membrane that molecule is 10000, working pressure 10kPa, and removing pigment, colloid and macromolecular substance, obtain ultrafiltration dialysis liquid;
3) electrodialysis: ultrafiltration dialysis liquid through continuous electrodialysis appts separating inorganic salts, working pressure 0.3Mpa, operating voltage 250V, electric current 3A;
4) reverse osmosis membrane concentrates: electric osmose liquid is the RO reverse osmosis membrane pre-concentration of 2 dusts through aperture, and working pressure 40kPa, obtains reverse osmosis transparent liquid and pure water, and production is returned in pure water recirculation;
5) concentrated, crystallization: be 30 (when 25 DEG C) by reverse osmosis transparent liquid vacuum concentration to degree Beaume, then crystallisation by cooling, collected by centrifugation crystallization, obtains a Cit crude product and crude product mother solution;
6) mother liquor is separated: have part Cit and a small amount of arginine and ornithine etc. in mother liquor, adopts ion exchange resin to be separated.With D001 cation exchange resin column on the flow velocity of 80 liters per hour, with 2M ammoniacal liquor wash-out, elutriant vacuum concentration to degree Beaume is 30 (when 25 DEG C), then crystallisation by cooling, and collected by centrifugation crystallization, obtains secondary Cit crude product;
7) crude product refining: by once with secondary Cit crude product mixing after add 100 times of weight pure water dissolve, then the activated carbon decolorizing accounting for solution weight 8% is added, filter, it is 30 (when 25 DEG C) that filtrate is concentrated into degree Beaume, crystallisation by cooling, collected by centrifugation crystallization, drying obtains Cit and refines sterling.
After testing, from reaction solution, be separated the product yield obtained is 91.5%, and Cit content is 99.4%.
Embodiment 5 produces Cit
1. transform arginine with the wet thallus being rich in arginine deiminase
Joined by wet thallus in the arginine aqueous solution, make arginine concentration of substrate be 10g/L, the concentration of wet thallus is 0.5g/L, and regulator solution pH is 7, under temperature 37 DEG C of conditions, react 0.5h.
After testing and calculate, the conversion of Arginine of 99.4% is Cit.
2. separation and purification Cit from reaction solution
1) micro-filtration: the ceramic microfiltration membrane by conversion fluid by aperture being 1 μm, working pressure 0.7kPa, obtains micro-filtration dialyzate;
2) ultrafiltration: micro-filtration dialyzate is 10 dusts by aperture, retains the PVDF ultrafiltration membrane that molecule is 2000, working pressure 50kPa, and removing pigment, colloid and macromolecular substance, obtain ultrafiltration dialysis liquid;
3) electrodialysis: ultrafiltration dialysis liquid through continuous electrodialysis appts separating inorganic salts, working pressure 0.05Mpa;
4) reverse osmosis membrane concentrates: electric osmose liquid is the RO reverse osmosis membrane pre-concentration of 6 dusts through aperture, and working pressure 90kPa, obtains reverse osmosis transparent liquid and pure water, and production is returned in pure water recirculation;
5) concentrated, crystallization: be 20 (when 25 DEG C) by reverse osmosis transparent liquid vacuum concentration to degree Beaume, then crystallisation by cooling, collected by centrifugation crystallization, obtains a Cit crude product and crude product mother solution;
6) mother liquor is separated: have part Cit and a small amount of arginine and ornithine etc. in mother liquor, adopts ion exchange resin to be separated.With D001 cation exchange resin column on the flow velocity of 100 liters per hour, with 5M ammoniacal liquor wash-out, elutriant vacuum concentration to degree Beaume is 20 (when 25 DEG C), then crystallisation by cooling, and collected by centrifugation crystallization, obtains secondary Cit crude product;
7) crude product refining: by once with secondary Cit crude product mixing after add 10 times of weight pure water dissolve, then the activated carbon decolorizing accounting for solution weight 5% is added, filter, it is 20 (when 25 DEG C) that filtrate is concentrated into degree Beaume, crystallisation by cooling, collected by centrifugation crystallization, drying obtains Cit and refines sterling.
After testing, from reaction solution, be separated the product yield obtained is 90.7%, and Cit content is 99.8%.
Claims (7)
1. derive from the arginine deiminase gene of bacillus cereus (Bacillus cereus) 93-11, its nucleotide sequence is as shown in SEQ ID NO:1, described bacillus cereus 93-11 is deposited in China typical culture collection center, and deposit number is CCTCC NO:M 2015047.
2. the recombination bacillus coli DH5 α I2-3 of arginine deiminase gene described in claim 1 is expressed in a strain, and be deposited in China typical culture collection center, deposit number is CCTCC NO:M2015048.
3. recombination bacillus coli according to claim 2 is producing the application in Cit.
4. a production method for Cit, is characterized in that comprising the following steps:
1) will China typical culture collection center be deposited in, deposit number be the recombination bacillus coli DH5 α I2-3 of CCTCC NO:M2015048 after seed culture and fermentation culture, the centrifugal wet thallus obtained containing arginine deiminase;
2) by step 1) wet thallus that obtains joins in the arginine aqueous solution, and regulate mixing solutions pH to be 6-7, under 37-60 DEG C of condition, react 0.5-4 hour;
3) separation and purification Cit from reaction solution.
5. the production method of Cit as claimed in claim 4, is characterized in that: step 2) described in mixing solutions arginic concentration be 10-300g/L, the concentration of wet thallus is 0.5-5g/L.
6. the production method of Cit as claimed in claim 4, is characterized in that: the method for described separation and purification Cit comprises the following steps:
1) micro-filtration: the ceramic microfiltration membrane by reaction solution by aperture being 0.02-1 μm, obtains micro-filtration dialyzate;
2) ultrafiltration: be 10-100 dust by aperture by micro-filtration dialyzate, retains the ultra-filtration membrane that molecule is 2000-10000, obtains ultrafiltration dialysis liquid;
3) electrodialysis: by ultrafiltration dialysis liquid through electrodialysis separating inorganic salts;
4) reverse osmosis membrane concentrates: by electrodialysis liquid through reverse osmosis membrane pre-concentration;
5) concentrated, crystallization: be 20-30, then crystallisation by cooling by reverse osmosis transparent liquid vacuum concentration to degree Beaume, collected by centrifugation crystallization, obtains a Cit crude product and mother liquor;
6) mother liquor is separated: by ion exchange resin column on mother liquor, with 2-5M ammoniacal liquor wash-out, elutriant vacuum concentration to degree Beaume is 20-30, then crystallisation by cooling, collected by centrifugation crystallization, obtains secondary Cit crude product;
7) crude product refining: by once with secondary Cit crude product mixing after add 10-100 times of weight pure water dissolve, then add activated carbon decolorizing, filter, it is 20-30 that filtrate is concentrated into degree Beaume, crystallisation by cooling, collected by centrifugation crystallization, drying obtains Cit and refines sterling.
7. the production method of Cit as claimed in claim 6, is characterized in that: described ion exchange resin column is D001 cation exchange resin column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510101168.6A CN104726478A (en) | 2015-03-09 | 2015-03-09 | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510101168.6A CN104726478A (en) | 2015-03-09 | 2015-03-09 | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104726478A true CN104726478A (en) | 2015-06-24 |
Family
ID=53450891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510101168.6A Pending CN104726478A (en) | 2015-03-09 | 2015-03-09 | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726478A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108299244A (en) * | 2018-01-31 | 2018-07-20 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of L-citrulline |
| CN108315364A (en) * | 2018-01-31 | 2018-07-24 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of α-ketoglutaric acid |
| WO2018133545A1 (en) * | 2017-01-23 | 2018-07-26 | 江南大学 | Gene engineering arginine deiminase reformed through site directed mutagenesis |
| CN108845070A (en) * | 2018-07-06 | 2018-11-20 | 精晶药业股份有限公司 | A kind of efficient liquid phase detection method of L-citrulline |
| 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 |
| CN113881724A (en) * | 2021-09-30 | 2022-01-04 | 新泰市佳禾生物科技有限公司 | Extraction and purification method for arginine-citrulline |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255944A (en) * | 1997-05-12 | 2000-06-07 | 凤凰药理学公司 | Modified arginine deiminase |
| WO2001083774A2 (en) * | 2000-05-04 | 2001-11-08 | Phoenix Pharmacologics, Inc. | Novel mutated form of arginine deiminase |
| CN101372465B (en) * | 2007-08-24 | 2011-11-16 | 北京健健康康生物技术有限公司 | Industrial method for extracting natural L-citrulline from water melon or smacking watermelon and other plant tissues |
| CN104212753A (en) * | 2013-05-31 | 2014-12-17 | 上海凯圣生物科技有限公司 | Arginine deiminase genetically engineered bacteria construction and purpose thereof |
-
2015
- 2015-03-09 CN CN201510101168.6A patent/CN104726478A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255944A (en) * | 1997-05-12 | 2000-06-07 | 凤凰药理学公司 | Modified arginine deiminase |
| WO2001083774A2 (en) * | 2000-05-04 | 2001-11-08 | Phoenix Pharmacologics, Inc. | Novel mutated form of arginine deiminase |
| CN101372465B (en) * | 2007-08-24 | 2011-11-16 | 北京健健康康生物技术有限公司 | Industrial method for extracting natural L-citrulline from water melon or smacking watermelon and other plant tissues |
| CN104212753A (en) * | 2013-05-31 | 2014-12-17 | 上海凯圣生物科技有限公司 | Arginine deiminase genetically engineered bacteria construction and purpose thereof |
Non-Patent Citations (3)
| Title |
|---|
| IVANOVA,N. ET AL.: "GenBank: AE016877.1,，REGION: 386655..387887", 《NCBI》 * |
| 张媛媛: "精氨酸脱亚胺酶的研究进展", 《中国酿造》 * |
| 李加友: "高产精氨酸脱亚胺酶菌株筛选鉴定和发酵特性研究", 《食品科技》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018133545A1 (en) * | 2017-01-23 | 2018-07-26 | 江南大学 | Gene engineering arginine deiminase reformed through site directed mutagenesis |
| US10829755B2 (en) | 2017-01-23 | 2020-11-10 | Jiangnan University | Genetically engineered arginine deiminase modified by site-directed mutagenesis |
| CN108299244A (en) * | 2018-01-31 | 2018-07-20 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of L-citrulline |
| CN108315364A (en) * | 2018-01-31 | 2018-07-24 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of α-ketoglutaric acid |
| CN108845070A (en) * | 2018-07-06 | 2018-11-20 | 精晶药业股份有限公司 | A kind of efficient liquid phase detection method of L-citrulline |
| 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 |
| CN113881724A (en) * | 2021-09-30 | 2022-01-04 | 新泰市佳禾生物科技有限公司 | Extraction and purification method for arginine-citrulline |
| CN113881724B (en) * | 2021-09-30 | 2024-08-16 | 新泰市佳禾生物科技有限公司 | Extraction and purification method for arginine-citrulline |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104726478A (en) | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli | |
| CN107502585A (en) | One plant of bacillus licheniformis engineering bacteria for efficiently synthesizing poly- γ glutamic acid | |
| CN107916283B (en) | A kind of production technology of niacinamide | |
| CN108467860B (en) | Method for high yield of gamma-aminobutyric acid | |
| CN111187759A (en) | Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by using enzyme method | |
| CN114836495B (en) | Construction and application of genetically engineered bacteria for producing NMN by nicotinamide fermentation | |
| CN109609582A (en) | A kind of method that microorganism catalysis deracemization prepares L-glufosinate-ammonium | |
| CN105238807A (en) | Construction of coenzyme efficient regeneration system and application thereof | |
| CN104152478A (en) | Method for coproducing D-arginine and gamatine through biotransformation | |
| CN108456652B (en) | Sphingosine monad gene engineering bacterium and construction method and application thereof | |
| CN108715827B (en) | Extracellular expression of tyrosine phenol lyase and application thereof | |
| CN104212757A (en) | Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli | |
| CN114854659B (en) | Ergothioneine production process and application thereof | |
| CN104152483A (en) | Application of argJ gene in fermentation production of L-citrulline | |
| CN101285085B (en) | Process for synthesizing adenosine methilanin by intact cell catalysis | |
| CN106119272B (en) | Strategy for efficiently co-producing L-phenylglycine and gluconic acid | |
| CN103146785B (en) | Production process for producing antiviral medicament ribavirin through bacillus amyloliquefaciens precursor addition fermentation method | |
| CN108424859B (en) | Construction and application of gene engineering bacteria for producing citicoline | |
| CN118064346B (en) | Recombinant genetic engineering strain, construction method thereof and fermentation production method of PQQ | |
| CN113881730B (en) | Method for synthesizing L-galactose | |
| CN114276970B (en) | Genetically engineered bacterium for producing 1, 3-propylene glycol | |
| CN116024278B (en) | Method for preparing D-pantothenic acid by fermentation method | |
| CN114875003B (en) | Mutant of short-chain dehydrogenase, coding gene obtaining method and application of mutant | |
| CN113308424B (en) | Bacillus pumilus for producing feruloyl esterase and application thereof | |
| CN112239730B (en) | Candida tropicalis mutant strain and application thereof in BCAA preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150624 |
|
| RJ01 | Rejection of invention patent application after publication |