CN103993031A - Preparation method for high-molecular-weight hyaluronic acid (HA), and engineering bacterium - Google Patents
Preparation method for high-molecular-weight hyaluronic acid (HA), and engineering bacterium Download PDFInfo
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Abstract
The invention relates to a preparation method for high-molecular-weight HA and an engineering bacterium. According to the method, a coding hyaluronic acid lyase gene (hylb) in the genome of Streptococcus zooepidemicus ATCC39920 is knocked out, and a hyaluronic acid lyase gene (hylb)-knocked out vector pSET4s:: hylb:: cmr is constructed. The method provided by the invention subjects a recombinant bacterium in which the hylb gene is knocked out to fermentation and analysis, the molecular weight of HA is measured by using an intrinsic viscosity process, and HA with a molecular weight of 3.9 * 10<6> Da is obtained.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to knocking out of hyaluronate lyase gene in streptococcus zooepidemicus genome, especially a kind of method and engineering bacteria of preparing high molecular weight hyaluronic acid.
Background technology
High molecular weight hyaluronic acid occupies an important position at makeup and medicine and other fields, and its preparation method is also difficult point and the focus of current biotechnology, for the preparation of the method for high molecular weight hyaluronic acid, is mainly animal extraction method.
Animal tissues's extraction method, hyaluronic acid (HA) is almost present in all animal tissuess, and the raw material that can be used in production is mainly people's umbilical cord, animal eyeball and cockscomb.Main technological process comprises that extraction, removal of impurities, enzymolysis, precipitation are with separated.This method mainly exists: raw material sources limitation is large, and product extraction yield is extremely low, and processing sequence is complicated, and production cost is high, may produce virus plant between the shortcoming such as cross infection.
Microbe fermentation method, the various bacteria of streptococcus has take the pod membrane that HA is main component, in growth and breeding process, to exocytosis, take the pod membrane that HA is main component, through separation and purification, gets final product to obtain high purity HA product.Compare with animal tissues extraction method, it is low that microbe fermentation method has cost, and industrial scale is not limited by animal material, and in fermented liquid, HA exists with unbound state, is easy to separation and purification and forms large-scale industrial produce the advantages such as the pathogenic bacterium pollution of animal origin-free.Along with the clone of important enzyme gene relevant to HA in microbial genome, building genetic engineering bacterium becomes the research and development focus that improves molecular weight.
Gene disruption is a kind of gene engineering, is a kind of of gene knockout (knockout) technology.But the sequence of Unknown Function known for certain sequence, at one section of resistant gene of the inner insertion of gene, make specific gene function forfeiture effect, thereby make partial function by barrier, and can further to organism, impact, and then infer the biological function that this gene.
Unidasa, the main left and right of performance in hyaluronic acid (HA) molecule enzymolysis process, it can cut HA molecule at random at HA intramolecule, generate HA small molecule segment, reduce the intermolecular interaction of HA, discharge bound water molecules, β-1 of degrading, the enzyme of 3 glycosidic links and β-Isosorbide-5-Nitrae glycosidic link remakes the small molecule segment for HA.Knock out hyaluronate lyase and can effectively suppress hyaluronic enzymolysis, thereby obtain the HA of high molecular.
Summary of the invention
The object of the present invention is to provide a kind of method and engineering bacteria of preparing high molecular weight hyaluronic acid, by building a kind of hyaluronate lyase gene hylb knockout carrier, knockout carrier is proceeded to Host Strains, by homologous recombination, make hylb gene inactivation, utilize resistance screening mark to obtain the engineering bacteria of hylb gene function disappearance, it is 3.9 * 10 that engineering bacterium fermentation is obtained to molecular weight
6the hyaluronic acid of Da.
The technical scheme that the present invention realizes object is as follows:
First the present invention take Streptococcus zooepidemicusATCC39920 strain gene group as the inner 1683bp gene fragment of template clone hyaluronate lyase gene hylb, with responsive to temperature type plasmid pSET4s (NCBI gene Bank:AB055650.1) for the carrier that sets out, insert the inner 1683bp gene fragment of hyaluronate lyase gene hylb, build intermediate carrier pSET4s::hylb, pSET1 (the NCBI gene Bank:AB042426.1) plasmid of take comprises the gene fragment that cmr mrna length is 1056bp as template amplification, this fragment is inserted into the DraIII site of hylb gene inside in intermediate carrier pSET4s::hylb, build knockout carrier pSET4s::hylb::cmr.Knockout carrier pSET4s::hylb::cmr electricity is proceeded to Streptococcus zooepidemicus ATCC39920 competent cell, utilize reconfiguring between homologous sequence DNA molecular, by comprising cmr mrna length, to be that the gene fragment of 1056bp will be inserted into hylb gene inner, make hylb gene inactivation, thereby obtain hylb gene knock-out bacterial strain.Knock-out bacterial strain is cultivated to 24h in 5L fermentor tank, get the fermented liquid of appropriate different time points, after processing, utilize limiting viscosity method to measure the molecular weight of HA, obtaining molecular weight is 3.9 * 10
6the hyaluronic acid of Da.
A kind of carrier that contains hyaluronate lyase gene, described carrier inserts the inner 1683bp gene fragment of hylb gene in initial carrier pSET4s, built intermediate carrier pSET4s::hylb, by one section, comprise the gene fragment that chloramphenicol resistance gene cmr length is 1056bp again and be inserted into the DraIII site in hylb gene fragment, form carrier pSET4s::hylb::cmr.
And the inner 1683bp gene fragment of described hylb gene starts from hylb gene start codon ATG downstream 202bp, ends at hylb gene terminator codon TAG upstream 1310bp, gene order is shown in sequence 1.
And described one section comprises the gene order that chloramphenicol resistance gene cmr length is 1056bp and sees sequence 2.
Carrier pSET4s::hylb::cmr proceeds to a Host Strains, and specificity knocks out hyaluronate lyase gene hylb, obtains the engineering bacteria of hylb gene function disappearance.
And the Host Strains of described engineering bacteria is streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC39920.
A method of preparing high molecular weight hyaluronic acid, is used above-mentioned engineering bacterium fermentation to obtain.
And it is 3.9 * 10 that described fermentation obtains hyaluronic molecular weight
6da.
And, the fermention medium of described engineering bacteria: sucrose 50g/L, yeast extract 3.5g/L, casein peptone 10g/L, NaCl1.5g/L, K2HPO42g/L, MgSO47H2O0.4g/L.
And, the fermentation condition of described engineering bacteria: 5L fermentor tank, liquid amount 3L, 8%, 37 ℃ of inoculum size, pH7.0, air flow 3vvm, fermentation 20-30 hour.
Advantage of the present invention and positively effect are as follows:
1, the present invention, from the angle of gene knockout, accurately locates, and directly knocking out the hyaluronate lyase gene acquisition product molecular weight relevant to hyaluronan molecule amount is 3.9 * 10
6the hyaluronic engineering bacteria of Da.
2, the present invention only knocks out hyaluronate lyase gene, does not affect the function of other genes.
Accompanying drawing explanation
Fig. 1 is knockout carrier pSET4s::hylb::cmr plasmid construction collection of illustrative plates of the present invention.
Fig. 2 is the homologous recombination schematic diagram of hylb gene knock-out bacterial strain screening of the present invention.
Fig. 3 is that the present invention proceeds to knockout carrier pSET4s::hylb::cmr electricity in the transformant screening figure of Streptococcus zooepidemicus ATCC39920 bacterial strain.
Fig. 4 is hylb gene knock-out bacterial strain PCR proof diagram, M:DNAmarker; 1:Streptococcus zooepidemicus ATCC39920; 2～3:hylb gene knock-out bacterial strain.
Fig. 5 is that hylb gene knock-out bacterial strain is at the molecular weight determination figure of the synthetic HA of different time points fermentation.
Embodiment
Below in conjunction with embodiment, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of carrier that contains hyaluronate lyase gene, by the inner 1683bp gene fragment of hylb gene, (this fragment originates in hylb gene start codon ATG downstream 202bp to described carrier, end at hylb gene terminator codon TAG upstream 1310bp) insert in initial carrier pSET4s (NCBI gene Bank:AB055650.1), built intermediate carrier pSET4s::hylb, by one section, comprise the gene fragment that chloramphenicol resistance gene cmr length is 1056bp again and be inserted into the DraIII site in hylb gene fragment, form carrier pSET4s::hylb::cmr.
Content of the present invention comprises: with responsive to temperature type plasmid pSET4s for the carrier that sets out, insert the inner 1683bp gene fragment of goal gene hylb gene, comprise cmr mrna length be the gene fragment of 1056bp build knock out plasmid pSET4s::hylb::cmr, produce the hylb gene knock-out bacterial strain of high molecular weight hyaluronic acid, the construction process of described hylb gene knock-out bacterial strain.
The inner 1683bp gene order of hylb gene of the present invention derives from streptococcus zooepidemicus (Streptococcus zooepidemicus), and bacterial strain deposit number is ATCC39920, and its nucleotide sequence is as shown in sequence 1.One section comprises the gene order that chloramphenicol resistance gene cmr length is 1056bp and derives from plasmid pSET1 (NCBI gene Bank:AB042426.1), and its nucleotide sequence is as shown in sequence 2.The described plasmid that knocks out inserts the inner 1683bp gene fragment of hylb gene in initial carrier pSET4s respectively, built intermediate carrier pSET4s::hylb, by one section, comprise the gene order that chloramphenicol resistance gene cmr length is 1056bp again and be inserted into the DraIII site in hylb gene fragment, be built into final knockout carrier pSET4s::hylb::cmr, and it is imported in streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC39920, the Streptococcus zooepidemicus ATCC39920 bacterial strain Streptococcus zooepidemicus/pSET4s::hylb::cmr that acquisition contains knockout carrier pSET4s::hylb::cmr.Above-mentioned bacterial strains, after gene recombination, obtains hylb gene knock-out bacterial strain.It is 3.9 * 10 that this knock-out bacterial strain obtains molecular weight after 5L fermentor cultivation
6the hyaluronic acid of Da.
The fermentation of hylb gene knock-out bacterial strain is produced HA molecular weight and is up to 3.9 * 10
6da, the synthetic hyaluronic method of its fermentation is: inoculation, in TSB liquid nutrient medium, through fermentation culture, is collected to fermented liquid, and through separation and purification, obtaining molecular weight is 3.9 * 10
6the hyaluronic acid of Da; Described culture temperature is 37 ℃; Incubation time 24h, pH value is 7.0.
The building process of concrete pSET4s::hylb::cmr is as follows:
One, the structure of hyaluronate lyase gene knockout carrier
The genome of streptococcus zooepidemicus (Streptococcus zooepidemicus ATCC39920) of take is template, at 202bp place, hylb gene start codon ATG downstream design primer hylb-F, at 1310bp place, hylb gene terminator codon TAG upstream design primer hylb-R, primer sequence is as shown in table 1, carries out the inner 1683bp gene fragment of pcr amplification hylb gene.
PCR reaction system is: 2 * PCR Buffer(is containing Mg
2+) 25 μ l, dNTP(25mM) 5 μ l, upstream primer hylb-F and downstream primer hylb-R(10 μ M) each 1 μ l, template (streptococcus zooepidemicus complete genome DNA) 1 μ l, KOD FxDNA polysaccharase (TOYOBO, KFX-101) 0.5 μ l, adding sterilized water to final volume is 50 μ l.
PCR reaction conditions is: 94 ℃ of denaturation 2min, and 98 ℃ of sex change 10s, 58 ℃ of annealing 30s, 68 ℃ are extended 2.5min, react 35 circulations, extend 10min after 68 ℃.
PCR finishes the complete object fragment nucleotide sequence of rear acquisition, and its sequence is shown in sequence 1.
Table 1 the primer sequence
Acquired DNA fragmentation is carried out to double digestion with PstI and BamHI, after reclaiming, be connected with the plasmid fragment pSET4s processing through same restriction endonuclease, to connect product and transform escherichia coli jm109 competent cell, and evenly coat on the LB flat board with spectinomycin resistance (50 μ g/ml), 37 ℃ of overnight incubation, picking mono-clonal, carries out bacterium colony PCR checking and enzyme and cuts checking, obtains carrier pSET4s::hylb.
PSET1 (the NCBI gene Bank:AB042426.1) plasmid of take is template, at 189bp place, chloramphenicol resistance gene terminator codon TAA downstream design primer cmr-R, at 216bp place, chloramphenicol resistance gene initiator codon ATG upstream design primer cmr-F, carry out one section of pcr amplification and comprise the gene fragment that cmr mrna length is 1056bp.
PCR reaction system is: 2 * PCRBuffer(is containing Mg
2+) 25 μ l, dNTP(25mM) 5 μ l, upstream primer cmr-F and downstream primer cmr-R(10 μ M) each 1 μ l, template (plasmid pSET1) 0.1 μ l, KOD Fx archaeal dna polymerase 0.5 μ l, adding sterilized water to final volume is 50 μ l.
PCR reaction conditions is: 94 ℃ of denaturation 2min, and 98 ℃ of sex change 10s, 58 ℃ of annealing 30s, 68 ℃ are extended 1min, react 35 circulations, extend 10min after 68 ℃.
PCR finishes the complete object fragment nucleotide sequence of rear acquisition, and its sequence is shown in sequence 2
Table 2 the primer sequence
The gene fragment of the acquired cmr of comprising gene is carried out to single endonuclease digestion with DraIII, after reclaiming, be connected with the plasmid fragment pSET4s::hylb processing through same restriction endonuclease, to connect product and transform escherichia coli jm109 competent cell, and evenly coat on the LB flat board with chlorampenicol resistant (20 μ g/ml), 37 ℃ of overnight incubation, picking mono-clonal, carries out bacterium colony PCR checking and enzyme and cuts checking, obtains knockout carrier pSET4s::hylb::cmr.
LB substratum:
Tryptones: 10.0g, yeast extract: 5.0g, NaCl:10.0g, after deionized water dissolving, is settled to 1.0L, and pH is adjusted to 7.0-7.2, and solid medium adds 1.5% agar powder.121 ℃ of sterilizing 20min.
Two, the structure of knock-out bacterial strain
The preparation of 2.1 streptococcus zooepidemicus competent cells
(1) from the streptococcus zooepidemicus glycerine pipe of-80 ℃ of preservations, draw in the liquid nutrient medium of 50uL bacterium liquid access 5mL THY and activate 24h, dilution 10 under 37 ℃ of 200rpm conditions
5-10
6doubly.Be coated on the flat board of THY, cultivate 24h for 37 ℃.
(2) the single bacterium colony of the above-mentioned cultivation 24h of picking access THY liquid nutrient medium in, under 37 ℃ of 200rpm conditions, cultivate 12h.
(3) the bacterium liquid of above-mentioned cultivation 12h is inoculated into by 1% inoculum size in the liquid nutrient medium of THY of fresh 100mL, under 37 ℃, 200rpm condition, be cultured to OD530 value and be about 0.37, the Unidasa adding (Shanghai Sheng Gong biotechnology company limited, 37326-33-3) 12.5ku, continue to cultivate 30min, cultivate OD530 value while finishing and be about 0.58.
(4) after cultivation finishes, by bacterium liquid ice bath 10min.
(5) use the aseptic centrifuge tube of 50ml by 12000rpm4 ℃ of centrifugal 10min of the bacterium liquid of above-mentioned precooling.
(6) after centrifugal, remove supernatant, add the resuspended thalline of the broad liquid liquid of sucrose 20ml of the 0.5M of ice bath.
(7) by resuspended liquid 12000rpm4 ℃ centrifugal 10min.
(8) supernatant discarded after centrifugal, with the ice-cold resuspended thalline of 0.5M sucrose solution of 5ml.
(9) by the resuspended liquid 12000rpm4 ℃ of centrifugal 10min of degree.
(10) supernatant discarded after centrifugal, adds after the resuspended thalline of sucrose solution of 500 μ L containing the 0.5M of 15% glycerine, and the resuspended liquid of every pipe 50 μ L divides and installs in the aseptic EP pipe of 1.5mL, and electricity turns or to put into-80 ℃ of refrigerators frozen immediately.
2.2 the electricity of streptococcus zooepidemicus competent cell transforms
(1) get 4ul and knock out plasmid pSET4s::hylb::cmr, join in the streptococcus zooepidemicus competent cell of the above-mentioned preparation of 50ul, fully mix.
(2) by above-mentioned, containing the competent cell that knocks out plasmid, transfer in the electric revolving cup of 2mm precooling, by following electroporation parameter, carry out electricity and transform:
(3) after electric shock, in electric revolving cup, add immediately the resuscitation fluid THY substratum of 1mL precooling, after fully mixing, transferred in aseptic EP pipe.
(4) until the centrifugal 3min of its 8000rpm after 30 ℃ of recovery 3h, collect thalline.
(5) thalline is coated in corresponding resistant panel, cultivated 48-76h for 30 ℃, transformant to be grown, taps into the single dual anti-secondary checking of row by transformant point.
(6) the correct clone of checking is inoculated in the THY liquid nutrient medium containing paraxin (20 μ g/mL), under 37 ℃ of 200rpm conditions, cultivate 12h, by its again streak inoculation to the flat board containing paraxin (20 μ g/mL) and spectinomycin (100 μ g/mL), verify the susceptibility to spectinomycin.
THY substratum:
Beef extract powder: 10.0g, Tryptones: 20.0g, glucose: 2.0g, yeast extract: 2.0g, NaHCO
3: 2.0g, NaCl:2.0g, Na2HPO4:0.4g, after deionized water dissolving, is settled to 1.0L, and pH is adjusted to 6.8, and solid medium adds 1.5% agar powder, 121 ℃ of sterilizing 20min.
The screening of 2.3hylb gene knock-out bacterial strain
(1) picking is verified correct transformant, is inoculated in the THY liquid nutrient medium that contains spectinomycin (100 μ g/mL) and paraxin (20 μ g/mL), cultivates 12h for 30 ℃.
(2) the bacterium liquid of above-mentioned cultivation 12h is seeded to not containing in the THY substratum of resistance by 1% inoculum size, cultivates 12h for 30 ℃.
(3) the bacterium liquid of above-mentioned cultivation 12h is seeded to not containing in the THY substratum of resistance by 1% inoculum size, cultivates 6～8h for 37 ℃.
(4) above-mentioned nutrient solution is pressed to decimal dilution method dilution 10
5-10
6doubly, be coated on the THY flat board that contains paraxin (20 μ g/mL), cultivate 24h for 37 ℃.
(5) mono-clonal obtaining in picking previous step is put respectively and is received dull and stereotyped (containing paraxin) of monoclonal antibody THY and dual anti-THY dull and stereotyped (containing spectinomycin and paraxin) is upper, 37 ℃ of cultivation 24h.
(6) repeat (3)-(5) step, until pick out hylb gene knock-out bacterial strain, i.e. growth and the bacterial strain of not growing on dual anti-flat board on monoclonal antibody flat board.
The checking of 2.4hylb gene knock-out bacterial strain
2.4.1hylb the PCR of gene knock-out bacterial strain checking
Extract the genome of above-mentioned recombinant bacterium, utilize the checking primer p1(in table 3 to be positioned at left arm primer hylb-F upstream 275bp), p2 (being positioned at right arm primer hylb-R downstream 250bp) carries out PCR checking, as successfully there is double exchange, chloramphenicol resistance gene can be inserted into goal gene inside, so just can amplify the fragment of 3273bp, as there is not double exchange, can amplify the fragment of 2208bp, as shown in Figure 3.
PCR reaction system is: 10 * PCR Buffer2 μ l, dNTP(10mM) 0.4 μ l, primer P1, P2(10 μ M) each 0.4 μ l, template 1 μ l, Taq archaeal dna polymerase (Beijing DingGuo ChangSheng Biology Technology Co., Ltd, PER001-1) 0.4 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 3min30s, react 35 circulations, extend 10min after 72 ℃.
Table 3 the primer sequence
Three, the Methanogenesis of hylb gene knock-out bacterial strain
Hylb gene knock-out bacterial strain ferments in 5L fermentor tank.Fermention medium used: sucrose 50g/L, yeast extract 3.5g/L, casein peptone 10g/L, NaCl1.5g/L, K
2hPO
42g/L, MgSO
47H
2o0.4g/L.Fermentation condition: 5L fermentor tank, liquid amount 3L, 8%, 37 ℃ of inoculum size, pH7.0, air flow 3vvm, fermentation 24h, regulates pH with 5MNaOH.Every 4h sampling once.
The detection of HA molecular weight: get the fermented liquid of appropriate different time points, add isopyknic 0.1%SDS to process 10min, by the centrifugal 15min for the treatment of solution 12000r/min, get the dehydrated alcohol precipitation at room temperature 30min of 3 times of volumes for supernatant.The centrifugal 10min of 12000r/min, abandon supernatant, to precipitate with ethylate solution washing once, fully dry under room temperature condition, then with appropriate 0.2MNaCl solution, fully dissolve, membrane filtration with 0.45 μ m, after filtration, with Ubbelohde viscometer, measure its limiting viscosity, with improvement carbazole method, measure HA content, the molecular weight that calculates HA according to following formula, precaution are as follows:
(1) 0.2M sodium chloride solution flows out the time t of Ubbelohde viscometer
0should be not less than 1.67mim.
(2) sample of different time points flows out the time t of Ubbelohde viscometer
1should be between t
01.3-1.5 doubly between.
(3) determine the HA content c of the sample of different time points.
(4) according to following formula, calculate limiting viscosity η, according to limiting viscosity, calculate molecular weight M η:
。
Claims (9)
1. a carrier that contains hyaluronate lyase gene, it is characterized in that: described carrier inserts the inner 1683bp gene fragment of hylb gene in initial carrier pSET4s, built intermediate carrier pSET4s::hylb, by one section, comprise the gene fragment that chloramphenicol resistance gene cmr length is 1056bp again and be inserted into the DraIII site in hylb gene fragment, form carrier pSET4s::hylb::cmr.
2. the carrier that contains hyaluronate lyase gene according to claim 1, it is characterized in that: the inner 1683bp gene fragment of described hylb gene starts from hylb gene start codon ATG downstream 202bp, end at hylb gene terminator codon TAG upstream 1310bp, gene order is shown in sequence 1.
3. the carrier that contains hyaluronate lyase gene according to claim 1, is characterized in that: described one section comprises the gene order that chloramphenicol resistance gene cmr length is 1056bp and sees sequence 2.
4. right 1 is required described carrier pSET4s::hylb::cmr to proceed to a Host Strains, specificity knocks out hyaluronate lyase gene hylb, obtains the engineering bacteria of hylb gene function disappearance.
5. engineering bacteria according to claim 4, is characterized in that: the Host Strains of described engineering bacteria is streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC39920.
6. a method of preparing high molecular weight hyaluronic acid, is characterized in that: right to use requires the engineering bacterium fermentation described in 4 or 5 to obtain.
7. the method for preparing high molecular weight hyaluronic acid according to claim 6, is characterized in that: it is 3.9 * 10 that described fermentation obtains hyaluronic molecular weight
6da.
8. the method for preparing high molecular weight hyaluronic acid according to claim 6, is characterized in that: the fermention medium of described engineering bacteria: sucrose 50g/L, yeast extract 3.5g/L, casein peptone 10g/L, NaCl1.5g/L, K2HPO42g/L, MgSO47H2O0.4g/L.
9. according to claim 6,7, one of the 8 described methods of preparing high molecular weight hyaluronic acid, it is characterized in that: the fermentation condition of described engineering bacteria: 5L fermentor tank, liquid amount 3L, 8%, 37 ℃ of inoculum size, pH7.0, air flow 3vvm, fermentation 20-30 hour.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184290A (en) * | 2019-05-21 | 2019-08-30 | 天津科技大学 | A kind of Genetic Recombination plasmid and engineering bacteria and application for producing macromolecule hyaluronic acid |
| CN112592930A (en) * | 2020-11-23 | 2021-04-02 | 天津科技大学 | Method and strain for improving hyaluronic acid yield |
| CN113881613A (en) * | 2021-04-13 | 2022-01-04 | 江南大学 | Method for producing and preparing hyaluronic acid with ultrahigh molecular weight by microbial fermentation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86100913A (en) * | 1985-01-18 | 1986-09-24 | 生物工艺总公司 | Method with producing high molecular weight sodium hyaluronate by fermentation of streptococcus |
| CN101294144A (en) * | 2008-01-25 | 2008-10-29 | 中国人民解放军第三军医大学 | Type II streptococcus suis sa1KR gene knockout mutant strain, preparation method and application thereof |
-
2013
- 2013-12-20 CN CN201310737032.5A patent/CN103993031B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86100913A (en) * | 1985-01-18 | 1986-09-24 | 生物工艺总公司 | Method with producing high molecular weight sodium hyaluronate by fermentation of streptococcus |
| CN101294144A (en) * | 2008-01-25 | 2008-10-29 | 中国人民解放军第三军医大学 | Type II streptococcus suis sa1KR gene knockout mutant strain, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SUN X 等: "Development of a markerless gene deletion system for Streptococcus zooepidemicus: functional characterization of hyaluronan synthase gene", 《APPL MICROBIOL BIOTECHNOL.》, vol. 97, no. 19, 31 October 2013 (2013-10-31), pages 8629 - 36, XP 035328602, DOI: doi:10.1007/s00253-013-5058-8 * |
| TAKAMATSU D 等: "Thermosensitive suicide vectors for gene replacement in Streptococcus suis", 《PLASMID》, vol. 46, no. 2, 30 September 2001 (2001-09-30), pages 140 - 8 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184290A (en) * | 2019-05-21 | 2019-08-30 | 天津科技大学 | A kind of Genetic Recombination plasmid and engineering bacteria and application for producing macromolecule hyaluronic acid |
| CN110184290B (en) * | 2019-05-21 | 2022-11-22 | 天津科技大学 | Genetic recombinant plasmid for producing high molecular weight hyaluronic acid, engineering bacterium and application |
| CN112592930A (en) * | 2020-11-23 | 2021-04-02 | 天津科技大学 | Method and strain for improving hyaluronic acid yield |
| CN112592930B (en) * | 2020-11-23 | 2023-03-10 | 天津科技大学 | Method and strain for improving hyaluronic acid yield |
| CN113881613A (en) * | 2021-04-13 | 2022-01-04 | 江南大学 | Method for producing and preparing hyaluronic acid with ultrahigh molecular weight by microbial fermentation method |
| CN113881613B (en) * | 2021-04-13 | 2023-07-25 | 江南大学 | Method for producing and preparing ultrahigh molecular weight hyaluronic acid by microbial fermentation method |
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