CN105112437A - Method for producing L-ornithine by aid of recombinant corynebacterium crenatum one-step fermentation process - Google Patents
Method for producing L-ornithine by aid of recombinant corynebacterium crenatum one-step fermentation process Download PDFInfo
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Abstract
The invention discloses a method for producing L-ornithine by the aid of a recombinant corynebacterium crenatum one-step fermentation process. The method includes that arginase from Bacillus cereus is successfully expressed in arginine high-yielding strains C. crenatum SDNN403 by the aid of shuttle plasmid pXMJ19 between Escherichia coli and corynebacterium crenatum. As shown by enzyme activity measurement results, original bacteria do not have the activity of the arginase, but the enzyme activity of the arginase of recombinant engineering bacteria can reach 24.3U/mg. Production of the L-ornithine by the aid of the one-step fermentation process and glucose which is a substrate is studied on the basis that engineering strains have characteristics of high-yielding arginine and the arginase. Owing to double-phase pH (potential of hydrogen) regulation strategies, totally 25.05g/L of L-arginine and 9.86g/L of the L-ornithine can be accumulated by the recombinant bacteria in 96h fermentation procedures at a first phase; totally 27.91g/L of L-ornithine is ultimately accumulated within 108h after fermentation at a second phase is carried out, but the content of the arginine is only 0.95g/L. The method has the advantages of high L-ornithine production efficiency, low L-arginine and other impurity acid content and easiness in separating and purifying a later-stage product, namely, the L-ornithine.
Description
Technical field
The clonal expression that the present invention relates to arginase gene obtains recombinant bacterial strain, this project bacterium is applied to the production of L-Orn simultaneously, belongs to biotechnology and biological technical field.
Technical background
Ornithine is a kind of basic aminoacids, and soluble in water and ethanol, is slightly soluble in the organic solvents such as ether.Ornithine accepts two molecule NH
4 +with a part CO
2form a part L-arginine.L-arginine can also be hydrolyzed into ornithine and urea under the effect of arginase.
L-Orn is a kind of important nonprotein amino acid, is the important precursor of arginine and proline synthesis.L-Orn is the important mesostate of ornithine cycle in human body simultaneously, and therefore it has important guaranteeing role to the function of detoxification of liver.L-Orn can stimulate the secretion of tumor growth hormone, promotes the synthesis of protein and the katabolism effect of carbohydrate and lipid.The effect of the vigor of liver under the amino acid that L-Orn is made together with other amino acid has good liver protecting and excites morbid state.Conbined usage L-Orn and toluylic acid effectively can treat hepatogenic encephalopathy, and particularly for the patient of urea cycle obstacle, L-Orn is by promoting that the synthesis of glutamine and excretion carry out stable reduction patient ammonia concentration.Ornithine alpha-ketoglutarate is good clinical nutrition agent, promotes the recovery of surgical wound patient, improves chronic malnutrition, improve immunologic function.Simultaneously ornithine and malate and Citrate trianion share the taste that can improve food and drink, reduce bitter taste.In Europe, the U.S. and Japan, L-Orn is all sold as Diet drugs.
The synthesis of industrial L-Orn comprises chemical synthesis, microbe fermentation method and L-arginine hydrolysis method.Chemosynthesis as the production method of early stage L-Orn, generally with propenal, prussic acid, ammonia, CO2 for Material synthesis L-Orn.But effect is not very desirable, main manifestations is the complex steps of synthesizing, and efficiency of pcr product is low, and the product obtained is the mixture of L-Orn and D-Orn, and the separation and purification for the later stage brings very large difficulty.Simultaneously chemical method synthesis L-Orn due to the hazardness of its environment, more and more not accept by modern industry.It is lower that biological synthesis process relative chemical synthesis method has production cost, advantages such as environmental pollution is little and coming into one's own.Producing L-ornithine by microorganism fermentation mainly obtains L-Orn superior strain by mutagenesis or engineered method, with the initial feed synthesis L-Orn the most such as glucose inexpensively or even starch.Japan's starting comparatively early in this respect, and the sixties in last century five just start the research that ornithine is produced, mainly based on mutagenesis screening.Nineteen fifty-seven kinoshita etc. first reported and utilizes the citrulline of corynebacterium glutamicum or arginic mutant strain fermentative production ornithine.After this, the people such as okumuraheShibuya have also been made a large amount of work summaries in this respect and have gone out to have the Corynebacterium glutamicum of arginine-deficient type and arginine hydroxamate resistance and had arginine and citrulline, and the citric acid Arthrobacter mutant strain of the mark such as mycophenolic acid is used in industrial production.The people such as domestic Chen Ning, Liu Shuqing are in the production of the ornithine that begins one's study in 1999, and be starting strain with Corynebacterium glutamicum, go out a kind of ornithine by ethyl sulfate and ultraviolet mutagenesis directive breeding and produce bacterium, and make ornithine output reach 9.85g/L by fermentation optimization.In recent years, along with the rise of metabolic engineering technology, by the method for metabolic engineering, the fermentative production of ornithine obtains certain progress, wherein, Zhongshan University Jiang Ling is gorgeous waits people to take Corynebacterium glutamicum as starting strain, by glutamate dehydrogenase and the glyceraldehyde-3-phosphate dehydrogenase of expressing heterologous, increase the content of NADPH in metabolic fluxes, L-Orn output is improved, most Zhongdao 14.8g/L, then Jiang is again by the method for metabolism evolution and metabolic engineering, and the output finally obtaining ornithine is 24.1g/L.Carry out ornithine production by fermentation method and can utilize better simply carbon source, as glucose etc., cost is very cheap, is suitable for industrial expansion.
CorynebacteriumcrenatumSDNN403 is the strain L-arginine superior strain that laboratory is obtained by multistage mutagenesis screening.On 5-L fermentor tank, glucose can be utilized in 96 hours to obtain the L-arginine (patent No. ZL03112896.3 of about 35/L, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCCNO.0890).L-arginine can be hydrolyzed and form L-Orn and urea under the effect of arginase.
The present invention is by expression vector pXMJ19, achieve and the arginase gene argI success deriving from Baciluscereus is expressed in C.crenatumSDNN403, and utilize this genetic engineering bacterium to ferment, take glucose as substrate, One-step production L-Orn, achieves the High-efficient Production of L-Orn.
Summary of the invention
Main research of the present invention: the present invention utilizes molecular engineering successfully to construct the engineering strain C.crenatumSDNN403/pXMJ19-argI of a plant height efficient expression arginase gene argI.Enzyme activity determination found that this recombinant bacterial strain has higher arginase activities.Based on the genetic engineering bacterium obtained, in 5-L fermentor tank, utilize the glucose method that two stage pH regulates and controls for substrate adopts to carry out L-Orn production to this bacterial strain probe into, and to fermentation condition, comprise induction time, MnSO
4addition, the opportunity etc. that two stage pH regulates and controls is optimized.Under 120g/L glucose adds, the accumulation volume obtaining L-Orn in 108h is 27.9g/L.
The technical scheme that the present invention takes:
The arginase gene argI deriving from bacterial strain Bacilluscereus is connected on expression vector pXMJ19, and is converted into C.crenatumSDNN403 and obtains recombinant bacterial strain.Based on this project bacterium, take glucose as substrate, the strategy also adopting two stage pH to regulate and control by fermentation method produces L-Orn.Fermentation condition is optimized simultaneously.
1. the structure of recombinant bacterial strain C.crenatumSDNN403/pXMJ19-argI
(1) according to argI gene order in Bacilluscereus full-length genome nucleotide sequence in NCBI, the PCR primer F of design arginase gene and R.
F：5’-ACCCG
AAGCTTATGAAAAAAGAAATCTCAGTTATTGG-3’(HindIII)
R：5’-ACCG
GGATCCTTATTTTAGTTTTTCACCGAATAAAG-3’(BamHI)
With Bacilluscereus genomic dna for template, the upstream and downstream primer amplification being arginase with F, R of designing obtains arginine gene fragment argI.The amplified fragments of acquisition is reclaimed by glue and to reclaim product after test kit reclaims in certain proportion and spend the night with pMD18-Tvector by a certain percentage to connect and separates, acquisition recombinant plasmid pMD18-T-argI.The recombinant vectors pMD18-T-argI of acquisition is delivered to Shanghai Sheng Gong biotech firm check order, after this recombinant plasmid and expression vector pXMJ19 being cut respectively through HindIII and BamHI enzyme after sequencing result is correct, reclaim corresponding fragment (argI fragment and enzyme cut after pXMJ19 fragment), then the fragment of recovery is connected under the effect of T4DNA ligase enzyme, obtain recombinant vectors pXMJ9-argI.This recombinant vectors pXMJ19-argI obtained is transferred in C.crenatumSDNN403 by electroporated method and obtains recombinant bacterium C.crenatumSDNN403/pXMJ19-argI.
2. recombinant bacterial strain enzyme activity determination
Enzyme activity determination method: preparation 0.2M substrate L-arginine (0.2M carbonate buffer solution, pH9.0), gets 0.9ml substrate solution, adds 0.1ml enzyme liquid, 40 DEG C of reaction 10min.Enzyme reaction solution is diluted corresponding multiple, gets the reaction solution after 1ml dilution, with the content of L-Orn in Chinard colorimetric method for determining reaction solution.The enzyme definition unit that lives becomes enzyme amount needed for L-Orn for 1min catalysis 1umolL-conversion of Arginine.
Chinard colorimetry: the Glacial acetic acid adding 1ml in 1ml reference liquid successively, 1ml mixing acid (ninhydrin solution), reacts 1h in boiling water bath, measures the absorbance under 515nm.
3. recombinant bacterium take glucose as the fermentation condition optimization that substrate one-step fermentation produces L-Orn
After the recombinant bacterium C.crenatumSDNN403/pXMJ19-argI of acquisition being transferred to LBG substratum (LB+0.5% glucose) activation culture twice, switching amount with 10% is transferred in 250mL shaking flask and is equipped with in the fermention medium of 25mL, 30 DEG C, 220rpm, carries out cultivation 96h in reciprocal shaker.
(1) induction time: the IPTG adding final concentration 0.7mM respectively at the different times of fermentation, carries out abduction delivering to the arginase of recombinant bacterium.Measure the output of L-arginine and L-Orn under different induction time
(2) MnSO
4addition: Mn
2+to arginase, there is stronger promoter action, in fermented liquid, add the Mn of different concns
2+l-Orn is produced on fermentation there is larger impact.The MnSO of different concns in design fermention medium
4addition.Different MnSO is measured after fermentation 96h
4the output of L-arginine and L-Orn under addition.
4. the two stage pH of recombinant bacterium regulates and controls fermentative Production L-Orn
Inoculum size with 4% after the recombinant bacterium C.crenatumSDNN403/pXMJ19-argI of acquisition being transferred to LBG substratum (LB+0.5% glucose) activation culture 16h is transferred in 250mL seed culture medium, at 30 DEG C, in 180r/min shaking table, cultivate 24h to OD
562be about 35.0.By seed with 10% inoculum size be transferred in 5-L fermentor tank, in 2.5L fermention medium, cultivate 96h.In the fermenting process of front 96h, fermented liquid pH is regulated by ammoniacal liquor, and maintains about 7.0.After 96h, the pH of fermented liquid is adjusted to about 9.0, L-Orn is produced in the fermentation carrying out subordinate phase, and maintains 12h.By the content of L-arginine, L-Orn and other related amino acids in Fermentation Liquor by High Performance Liquid Chromatography.
(1) seed culture medium (g/L): glucose, 60; Yeast powder, 10; (NH4)
2sO
4, 2; KH
2pO
4, 2.7; K
2hPO4,0.5; MgSO
47H
2o, 0.2.
(2) fermention medium (g/L): glucose, 120; Yeast powder, 8; KH
2pO
4, 27; K
2hPO4,0.5; MnSO
4h
2o, 0.5; MgSO
47H
2o, 0.2;
(3) amino acid analysis chromatographic condition
(a) chromatographic column: Agilent chromatographic column TC-C18250*4.6*5
(b) column temperature: 40 DEG C
(C) moving phase: A phase: take 8.0 grams of crystallization sodium acetates in 1000 ml beakers, adds 1000 ml waters and is stirred to all crystal water and dissolves, then add 225 microlitre triethylamines, stir and drip 5% acetic acid, PH is transferred to 7.20 ± 0.05; Add 5 milliliters of tetrahydrofuran (THF)s, for subsequent use after mixing.B phase: take 12.0 grams of crystallization sodium acetates in 800 ml beakers; Add 400 ml waters and be stirred to all dissolving crystallizeds; Drip 5% acetic acid and PH is transferred to 7.20 ± 0.05; This solution is added 800 milliliters of acetonitriles and 800 ml methanol, for subsequent use after mixing.
(d) detector: UV-detector 338nm
Advantage of the present invention and positively effect are:
(1) the present invention with high yield arginine bacterial strain C.crenatumSDNN403 for starting strain, build the recombinant corynebacterium crematum SDNN403 with efficient arginase activities, without the need to expensive L-arginine for raw material, can directly utilize cheap glucose to carry out the production of L-Orn for raw material.Thus effectively reduce the production cost of L-Orn.
(2) optimal reaction pH with C.crenatumSDNN403 that the present invention is based on arginase ferments, and to produce pH needed for L-arginine different and take the strategy of two stage pH regulation and control, the pH of fermented liquid is maintained about 7.0 by the first stage, makes recombinant bacterium can be that substrate produces L-arginine with glucose; Second valency section, by about fermented liquid pH regulator to 9.0, makes arginine play the character of its optimal conversion L-arginine enzyme.Experiment proves the strategy that the two stage pH of employing regulates and controls, and makes fermentation produce survey L-arginine and substantially can change into L-Orn.The L-Orn of 27.9g/L is run up in fermented liquid.
Embodiment
Embodiment 1: the structure of recombinant bacterium C.crenatumSDNN403/pXMJ19-argI
(1) according to argI gene order in Bacilluscereus full-length genome nucleotide sequence in NCBI, the PCR primer F of design arginase gene and R.
F：5’-ACCCG
AAGCTTATGAAAAAAGAAATCTCAGTTATTGG-3’(HindIII)
R：5’-ACCG
GGATCCTTATTTTAGTTTTTCACCGAATAAAG-3’(BamHI)
(2) clone of arginase gene
With Bacilluscereus STb gene for template, the primer that utilization provides above does pcr amplification, and amplification condition is: 94 DEG C of denaturations, 5min, a circulation; 94 DEG C of sex change, 1min, 56 DEG C of annealing, 1min, 72 DEG C of extensions, 45s, 35 circulations; 72 DEG C of ends extend 10min.PCR amplification system: template 1 μ L, upstream and downstream primer each 0.4 μ L, dNTPMix4 μ L, 10 × ExTaqBuffer5 μ L, the distilled water 37 μ L of sterilizing, ExTaqDNA polysaccharase 1 μ L.Adopt gel to reclaim test kit and carry out purifying and recovery to PCR primer, the concentration of product is reclaimed in electrophoresis inspection.Reclaiming product leaves in the centrifuge tube of 1.5mL, and-20 DEG C of Refrigerator stores are for subsequent use.Reclaim product to be connected with pMD18-TVector, connect product conversion E.coilJM109, converted product coating is dull and stereotyped containing the LB of penbritin, through 37 DEG C of overnight incubation, picking colony is in 10mL LB liquid medium, and 37 DEG C of incubator overnight extract plasmid after cultivating, called after pMD18-T-argI, after digestion verification successful connection, add glycerine to final concentration 15% ~ 20% (w/v) ,-70 DEG C of Storage in refrigerator.
(3) structure of recombinant plasmid pXMJ19-argI
Extract plasmid pMD18-T-argI and pXMJ19 be stored in E.coliJM109, and carry out double digestion with BamHI and HindIII respectively, connect after utilizing gel to reclaim test kit recovery, linked system: goal gene digestion products 7 μ L, pXMJ19 digestion products 1 μ L, T4DNA ligase enzyme buffer1 μ L, T4DNA ligase enzyme 1 μ L, 16 DEG C of connections of spending the night.The recombinant plasmid pXMJ9-argI connected is transformed into competence E.coilJM109, with LBG (LB+0.5%Glc) chlorampenicol resistant substratum, the positive bacterium colony of picking.37 DEG C of incubator overnight extract plasmid, called after pXMJ9-argI after cultivating, and after digestion verification is correct, add glycerine to final concentration 15% ~ 20% (w/v) ,-70 DEG C of Storage in refrigerator are for subsequent use.
(4) recombinant plasmid pXMJ9-argI transforms C.crenatumSDNN403
Prepared by competence: picking C.crenatumSDNN403 is inoculated in a 10mLLBG (LB+0.5% glucose) liquid nutrient medium, 30 DEG C of shaking table overnight incubation, the bacterium liquid getting 500 μ L incubated overnight is transferred containing in the 50mL LB liquid medium of 3% glycine and 0.1% tween-80, makes initial cell OD
600reach 0.3 in 30 DEG C, 200r/min cultivates cell OD
600reach 0.9.By bacterium liquid precooling 15min after cell cultures terminates, then collected by centrifugation thalline.Wash thalline 4 times with 10% glycerine of precooling, finally use 0.2mL10% glycerine re-suspended cell, with the packing of 1.5mL pipe, often pipe 80ul is directly used in electricity conversion.
Electricity turns: 1800V electricity turns 5ms, and electricity adds 800ulLB substratum 30 DEG C and cultivates 2-3h after turning
Recombinant bacterium obtains: coat chlorampenicol resistant LBG substratum, 30 DEG C of cultivations, and the positive bacterium colony of picking, extracts plasmid enzyme restriction checking, obtain recombinant bacterium C.crenatumSDNN403/pXMJ19-argI.
Embodiment 2: recombinant bacterium C.crenatumSDNN403/pXMJ19-argI arginase vitality test
The recombinant bacterium C.crenatumSDNN403/pXMJ19-argI that embodiment 1 is built, 10mL is inoculated in respectively containing in the LBG substratum of paraxin with starting strain C.crenatumSDNN403,30 DEG C of shaking culture are spent the night, next day by 1% inoculum size transfer in 50mLLBG substratum, cultivate after 8h to OD for 30 DEG C
600add the IPTG that final concentration is 0.7mM when being 0.9,30 DEG C of induction 8h, get fermented liquid in 4 DEG C, the Tris-HCl buffer solution for cleaning of 10000r/min centrifugal 10min, pH7.0 3 times, finally uses in 5mLpH7.0Tris-HCl damping fluid.Crude enzyme liquid is prepared in ultrasonic disruption process.
Preparation 0.2M substrate L-arginine (pH100.2M carbonate buffer solution), gets 0.9ml substrate solution, adds 0.1ml enzyme liquid, 40 DEG C of reaction 10min.Enzyme reaction solution is diluted corresponding multiple, gets the reaction solution after 1ml dilution, with the content of L-Orn in Chinard colorimetric method for determining reaction solution.The enzyme definition unit that lives becomes enzyme amount needed for L-Orn for 1min catalysis 1umolL-conversion of Arginine.
Chinard colorimetry: the Glacial acetic acid adding 1ml in 1ml reference liquid successively, 1ml mixing acid (ninhydrin solution), reacts 1h in boiling water bath, measures the absorbance under 515nm.
Result show arginase that recombinant bacterium C.crenatumSDNN403/pXMJ19-argI expresses more alive than enzyme be 24.3U/mg, and original bacteria C.crenatumSDNN403 does not detect that arginic enzyme is lived, thus achieve growing out of nothing of in C.crenatumSDNN403 arginase.
Embodiment 3: recombinant bacterium C.crenatumSDNN403/pXMJ19-argI fermentation method produces the condition optimizing of L-Orn
After the recombinant bacterium C.crenatumSDNN403/pXMJ19-argI of acquisition being transferred to LBG substratum (LB+0.5% glucose) activation culture twice, switching amount with 10% is transferred in 250mL shaking flask and is equipped with in the fermention medium of 25mL, 30 DEG C, 220rpm, carries out cultivation 96h in reciprocal shaker.
(1) induction time optimization: the IPTG being 0.6mM at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h interpolation final concentration respectively carries out abduction delivering to the arginase of recombinant bacterium.
Result shows, IPTG is at earlier fermentation (0h, 12h and 24h) when adding, the L-arginine that fermentation produces can change into L-Orn substantially, particularly when 0h and 12h interpolation IPTG induces, L-arginine content in fermented liquid only has about 0.5g/L, and when not adding IPTG, the L-arginine content in fermented liquid is up to about 22.5g/L.But the acid production rate that causes too early of the interpolation time of IPTG significantly reduces, when 0h or 12h interpolation IPTG induces, the comprehensive acid production rate of L-arginine and L-Orn only has about 15%.And phase (84h) or when not adding IPTG, the comprehensive acid production rate of L-arginine and L-Orn reaches 29.6% after fermentation.Consider the factor of two aspects, show that 24h is best induction starting time.
(2) MnSO
4the interpolation time: add 0mM in the fermentation medium respectively, the MnSO of 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM and 2.0mM
4
Result shows, MnSO
4the output of addition L-Orn within the scope of 0-0.14mM substantially along with MnSO
4the rising of concentration and increasing, increases MnSO further
4addition can not increase the output of L-Orn.Its reason is Mn
2+the enzyme of promotion arginase that can be stronger is lived, certain density Mn
2+the enzyme that can improve arginase is lived, and makes L-arginine be converted into L-Orn, improves the output of L-Orn.Comprehensively show, MnSO
4the best add concentration be 0.14mM.
Embodiment 4: the two stage pH of recombinant bacterium C.crenatumSDNN403/pXMJ19-argI regulates and controls fermentative Production L-Orn
Inoculum size with 4% after the recombinant bacterium C.crenatumSDNN403/pXMJ19-argI of acquisition being transferred to LBG substratum (LB+0.5% glucose) activation culture 16h is transferred in 250mL seed culture medium, at 30 DEG C, in 180r/min shaking table, cultivate 24h to OD
562be about 35.0.By seed with 10% inoculum size be transferred in 5-L fermentor tank, in 2.5L fermention medium, cultivate 96h.Adding final concentration when 24h is that the IPTG of 0.6mM induces.In the fermenting process of front 96h, fermented liquid pH is regulated by ammoniacal liquor, and maintains about 7.0.After 96h, the pH of fermented liquid is adjusted to about 9.0, L-Orn is produced in the fermentation carrying out subordinate phase, and maintains 12h.By the content of L-arginine, L-Orn and other related amino acids in Fermentation Liquor by High Performance Liquid Chromatography.
Result shows, recombinant bacterium C.crenatumSDNN403/pXMJ19-argI is in the fermentation of first stage, and codeposition tires out the L-arginine of 25.05g/L and the L-Orn of 9.86g/L.After the fermentation of second stage, finally obtain the L-Orn of 27.91g/L, and arginic content is only 0.95g/L.
Claims (5)
1. a structure for novel recombinant expression vector, is characterized in that with Bacilluscereus genomic dna for template, and amplification obtains arginine gene argI, is cloned on expression vector pXMJ19, builds recombinant expressed type plasmid pXMJ19-argI.
2. a strain has the recombination engineering bacteria of living compared with homoarginine enzyme enzyme, it is characterized in that the recombinant vectors pXMJ19-argI electric shock conversion method built in claim 1 to be transformed in C.crenatumSDNN403, and argI can express efficiently under IPTG induction.
3. the genetic engineering bacterium C.crenatumSDNN403 that homoarginine enzyme enzyme according to claim 2 is lived is applied to the method that one-step fermentation produces L-Orn, it is characterized in that, need using the high L-arginine of price as substrate, recombinant bacterium C.crenatumSDNN403/pXMJ19-argI directly utilizes cheap glucose to carry out fermentative production L-Orn for substrate.
4. application of policies according to claim 3 produces the method in L-Orn in one-step fermentation, it is characterized in that: the inoculum size with 4% after recombinant bacterium is transferred to LBG substratum (LB+0.5% glucose) activation culture 16h is transferred in 250ml seed culture medium, at 30 DEG C, in 180r/min shaking table, cultivate 24h to OD
562be about 35.0; By seed with 10% inoculum size be transferred in 5-L fermentor tank, in 2.5L fermention medium, cultivate 96h; Adding final concentration when 24h is that the IPTG of 0.6mM induces; In the fermenting process of front 96h, fermented liquid pH is regulated by ammoniacal liquor, and maintains about 7.0; After 96h, the pH of fermented liquid is adjusted to about 9.0, L-Orn is produced in the fermentation carrying out subordinate phase, and maintains 12h.
5. seed culture medium according to claim 4 and fermention medium component, is characterized in that, seed culture medium (g/L): glucose 60, yeast powder 10, (NH4)
2sO
42, KH
2pO
42.7, K
2hPO40.5, MgSO
47H
2o0.2; Fermention medium (g/L): glucose 120, yeast powder 8, KH
2pO
427, K
2hPO40.5, MnSO
4h
2o0.5, MgSO
47H
2o0.2.
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| CN106282085A (en) * | 2016-11-08 | 2017-01-04 | 江南大学 | A kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin |
| CN106544311A (en) * | 2016-11-08 | 2017-03-29 | 江南大学 | Synthesize acetoin and lactic acid using Corynebacterium crenatum resting cell glucose |
| CN108893438A (en) * | 2018-06-25 | 2018-11-27 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-Orn yield |
| CN114277070A (en) * | 2021-11-29 | 2022-04-05 | 新泰市佳禾生物科技有限公司 | Method for producing L-ornithine by fermentation |
| CN114277070B (en) * | 2021-11-29 | 2024-07-09 | 新泰市佳禾生物科技有限公司 | Method for producing L-ornithine by fermentation |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441055A (en) * | 2003-02-25 | 2003-09-10 | 江南大学 | L-arginine producing strain and its mutation method and usage in producing L-arginine |
| CN101955901A (en) * | 2010-09-19 | 2011-01-26 | 南京工业大学 | Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same |
| CN102234624A (en) * | 2011-04-25 | 2011-11-09 | 武汉远大弘元股份有限公司 | Genetic engineering strain for expression and generation of bacillus subtilis arginase, and constructing method thereof |
| CN102433288A (en) * | 2012-01-16 | 2012-05-02 | 江南大学 | Strain for producing ornithine and method for biologically synthesizing ornithine with same |
| US20130309733A1 (en) * | 2012-05-21 | 2013-11-21 | Cathay R&D Center Co., Ltd. | Stabilized recombinant expression plasmid vector in hafnia alvei and applications thereof |
| CN103923936A (en) * | 2013-12-05 | 2014-07-16 | 江南大学 | Construction of arginase production engineering bacteria and application of arginase production engineering bacteria in production of L-ornithine |
| CN103966151A (en) * | 2013-01-29 | 2014-08-06 | 上海凯圣生物科技有限公司 | Construction method and applications of genetic engineering bacterium for producing L-arginine |
| CN104031933A (en) * | 2014-05-08 | 2014-09-10 | 江南大学 | Construction and application method for L-ornithine synthesis bacteria |
| CN104830922A (en) * | 2015-06-01 | 2015-08-12 | 南京大学 | Enzymatic conversion method of L-ornithine |
-
2015
- 2015-08-25 CN CN201510529068.3A patent/CN105112437A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441055A (en) * | 2003-02-25 | 2003-09-10 | 江南大学 | L-arginine producing strain and its mutation method and usage in producing L-arginine |
| CN101955901A (en) * | 2010-09-19 | 2011-01-26 | 南京工业大学 | Corynebacterium glutamicum and method for preparing L-ornithine and salts thereof by using same |
| CN102234624A (en) * | 2011-04-25 | 2011-11-09 | 武汉远大弘元股份有限公司 | Genetic engineering strain for expression and generation of bacillus subtilis arginase, and constructing method thereof |
| CN102433288A (en) * | 2012-01-16 | 2012-05-02 | 江南大学 | Strain for producing ornithine and method for biologically synthesizing ornithine with same |
| US20130309733A1 (en) * | 2012-05-21 | 2013-11-21 | Cathay R&D Center Co., Ltd. | Stabilized recombinant expression plasmid vector in hafnia alvei and applications thereof |
| CN103966151A (en) * | 2013-01-29 | 2014-08-06 | 上海凯圣生物科技有限公司 | Construction method and applications of genetic engineering bacterium for producing L-arginine |
| CN103923936A (en) * | 2013-12-05 | 2014-07-16 | 江南大学 | Construction of arginase production engineering bacteria and application of arginase production engineering bacteria in production of L-ornithine |
| CN104031933A (en) * | 2014-05-08 | 2014-09-10 | 江南大学 | Construction and application method for L-ornithine synthesis bacteria |
| CN104830922A (en) * | 2015-06-01 | 2015-08-12 | 南京大学 | Enzymatic conversion method of L-ornithine |
Non-Patent Citations (7)
| Title |
|---|
| CHEN XUE LAN等: "Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum", 《BIOMED ENVIRON SCI》 * |
| MEIJUAN XU等: "The Role of ARGR Repressor Regulation on L-arginine Production in Corynebacterium crenatum", 《APPL BIOCHEM BIOTECHNOL》 * |
| WEI SONG等: "Enzymatic production of l-ornithine from l-arginine withrecombinant thermophilic arginase", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| WENFANG DOU等: "Improvement of L-Arginine Production by Overexpression of a Bifunctional Ornithine Acetyltransferase in Corynebacterium crenatum", 《APPL BIOCHEM BIOTECHNOL》 * |
| 方建军等: "微生物法生产鸟氨酸的代谢工程研究进展", 《中国食品工业》 * |
| 赵芹芹等: "钝齿棒杆菌的代谢改造：L-鸟氨酸与L-瓜氨酸合成菌株的构建", 《微生物学报》 * |
| 黄仲华主编: "《中国调味食品技术实用手册》", 28 February 1991 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282085A (en) * | 2016-11-08 | 2017-01-04 | 江南大学 | A kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin |
| CN106544311A (en) * | 2016-11-08 | 2017-03-29 | 江南大学 | Synthesize acetoin and lactic acid using Corynebacterium crenatum resting cell glucose |
| CN108893438A (en) * | 2018-06-25 | 2018-11-27 | 江南大学 | A method of it improving Corynebacterium crenatum and synthesizes L-Orn yield |
| CN114277070A (en) * | 2021-11-29 | 2022-04-05 | 新泰市佳禾生物科技有限公司 | Method for producing L-ornithine by fermentation |
| CN114277070B (en) * | 2021-11-29 | 2024-07-09 | 新泰市佳禾生物科技有限公司 | Method for producing L-ornithine by fermentation |
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