CN1587387A - Method for producing hyaluronic acid and its special strain - Google Patents
Method for producing hyaluronic acid and its special strain Download PDFInfo
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- CN1587387A CN1587387A CN 200410070768 CN200410070768A CN1587387A CN 1587387 A CN1587387 A CN 1587387A CN 200410070768 CN200410070768 CN 200410070768 CN 200410070768 A CN200410070768 A CN 200410070768A CN 1587387 A CN1587387 A CN 1587387A
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- suis
- streptococcus
- hyaluronic acid
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- vgb
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 18
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 18
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 18
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 101150112266 vgb gene Proteins 0.000 claims abstract description 19
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- 238000000034 method Methods 0.000 claims description 19
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- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
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- 241000607626 Vibrio cholerae Species 0.000 claims description 5
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- 229910052760 oxygen Inorganic materials 0.000 abstract description 13
- 239000001301 oxygen Substances 0.000 abstract description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 4
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
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- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The production process with high hyaluronic acid yield includes constructing a plasmid expressing vector containing vgb gene sequence, and introducing into streptococcus for expression. The constructed plasmid expressing vector pEUVGB is obtained through inserting the gene sequence vgb into the polyclonal site of plasmid vector pEU308. The streptococcus is preferably epizootic streptococcus. By means of expressing vgb gene in epizootic streptococcus, the present invention can raise the oxygen utilization of thallus to promote thallus growth and increase the yield of hyaluronic acid. The present invention is used in the industrial fermentation of producing hyaluronic acid, and can ensure oxygen supply and raise the yield of hyaluronic acid.
Description
Technical field
The present invention relates to produce hyaluronic method and special strain therefore thereof.
Background technology
Hyaluronic acid (hyaluronic acid, HA), have another name called glass acid, be Meyer and Palmer in 1934 isolated a kind of high viscosity substance (Meyer K. from bovine vitreous body at first, Palmer J.W.The polysaccharideof the vitreous humor.J.biol.chem, 1934,107:629-634).After this, people have carried out a large amount of research to distribution, chemical structure, physiological function and the clinical application of HA again, particularly Balazs is used for HA complicated surgery of retinal detachment first in nineteen sixty, and obtained after the ideal curative effect, the relevant research of HA in the medical applications field has obtained a series of great progress again.HA is as a kind of biochemical drug, not only medically be widely used, in the day chemical industry, because HA has the good moisture preserving effect, be again the natural biological molecule that extensively exists in skin and other tissue, be used for makeup, be described as ideal natural moisturizing factor (Ling Peixue since the eighties in 20th century, He Yanli, Guo Xueping.Hyaluronic acid.China Light Industry Press, 2000), thereby it also has broad application prospects in cosmetic industry.
The initial production method of HA is to extract from animal tissues's (mainly being cockscomb, buphthalmos).This animal tissues extraction method cost height, HA content low (the general fresh cockscomb of 1kg only can extract 4gHA), and separation and purification complexity, the molecular weight of the HA of extraction is also smaller.Nineteen thirty-seven, discovery Hemolytic streptococcus streptococcushaemolyticus such as Kendall can synthesize the hyaluronic acid justacrine and form pod membrane (Kendall F.E.Heidelberger M.Dawson M.H.A serologically inactive polysaccharide elaborated by mucoid strain ofgroup A hemolytic Streptococcus.J.Biol.Chem.1937,118 (1): 61-69) outside born of the same parents; Again successively reported the microbial strains that can synthesize HA thereafter.Because it is the pod membrane of unique composition that many suis can be synthesized with the HA polysaccharide, make suis (particularly to the less C type of human body harm) become the maximum HA of research and produce bacterium, that has reported can have as the suis that HA produces bacterium: Hemolytic streptococcus (A type), streptococcus zooepidemicus (C type), streptococcus equi (C type), excrement streptococcus equi (C type), hypogalactia suis (C type) and bacillus cholerae gallinarum (C type) etc.; Wherein the A type is the human pathogen, and the C type then can only infect domestic animals.Nineteen eighty-three, reported first Japanese Shiseido produce HA with suis (red slope day engage in this profession, the rugged good fortune of a specified duration of coltfoal, Liu Guangnan, Shiseido.ア Le ロ Application acid manufacture method.Day disclosure special permission communique, clear 58-56692,1983), subsequently, states such as Great Britain and America have reported the method that adopts Production by Microorganism Fermentation HA in succession, the source of having widened HA has greatly promoted research and the application of HA.The realization that microbial fermentation is produced HA is the most great progress that the HA production field is being obtained in recent years.Streptococcus can be grown under aerobic and oxygen free condition in the common reed anerobe.And from the angle of metabolic regulation, aerobic fermentation helps synthetic (Goh, the L.T.Fermentation studies of hyaluronic acidfermentat ion by Streptococcus zooepidemicus.In Chemical Engineering of HA; Brisbane:University of Queensland, 1998).But because the hyaluronic acid that produces is wrapped in around the thalline, increased the fermented liquid viscosity during the fermentation, reduced dissolved oxygen, limited growth and the hyaluronic output of bacterium, become an important factor of restriction hyaluronic acid fermentation industry.
Vitreoscilla (Vitreoscilla sp.) is the aerobic Gram-negative bacteria of a kind of obligate, in low-oxygen environment, induce synthetic Vitreoscilla hemoglobin (Vitreoscilla hemoglobin, VHb), its encoding gene is Vitreoscilla hemoglobin gene (Vitreoscilla hemoglobin gene, vgb, sequence is seen the sequence 1 in the sequence table), this oxyphorase can be induced synthetic under extremely low dissolved oxygen amount (＜5%) in a large number, make Vitreoscilla (as the rotten ground of mire) existence (Fish P A well in the oxygen deprivation environment, Webster D A, Stark B C.Vitreoscillahemoglobin enhances the first step in 2,4-dinitroluene degradation in vitroand at low aeration in vivo, Journal of Molecular Catalysis B:Enzymatic, 2000,9:75-82).VHb participates in the metabolism relevant with oxygen with the oxygenate attitude, by oxygen is passed to respiratory chain, and regulates the activity of terminal oxidase, changes the efficient of oxidative phosphorylation, and then changes the pathways metabolism under the hypoxia condition, down to having influence on some expression of gene.It can improve the reorganization bacterium from molecular level oxygen utilized ability (Jacobsen J R, Khosla C.New directions in metabolic engineering.CurrentOpinion in Chemical Biology 1998,2:133-137).Some reported in literature the application example of vgb gene, show that it has and promote cell growth, the effect that improves cell culture density and exogenous gene expression.Can be as the expression of vgb gene in streptomyces aureus with synthetic raising 40%～60% (first month of spring, Ye Qin, Shi Xianai, Qiu Li, Song Siyang, the Guo Yanghao of product Streptomycin sulphate.The cloning and expression of Vitreoscilla hemoglobin gene in streptomyces aureus.The microorganism journal, 2002,42 (3): 305-310).
Summary of the invention
The purpose of this invention is to provide hyaluronic method of a kind of production and special strain therefore thereof.
The hyaluronic special strain therefore of production provided by the present invention is the reorganization suis that contains the vgb gene.
Described suis is any one in Hemolytic streptococcus, streptococcus zooepidemicus, streptococcus equi, excrement streptococcus equi, hypogalactia suis and the bacillus cholerae gallinarum.
Described suis is preferably any one in A type Hemolytic streptococcus, C typothere epidemic disease suis, C type streptococcus equi, C type excrement streptococcus equi, C type hypogalactia suis and the C type bacillus cholerae gallinarum.
Described suis is streptococcus zooepidemicus ATCC 39920 more preferably.
The hyaluronic method of production provided by the present invention is to make up the plasmid expression vector that contains the vgb gene order, and the carrier that makes up is imported in the suis, obtains the reorganization suis, and fermentation reorganization suis obtains hyaluronic acid.
The pEUVGB that the constructed plasmid expression vector obtains for the multiple clone site that the vgb gene is inserted into carrier pEU308 (Eichenbaum Z., FederleM.J.1998.Use of the lactococcal nisA promoter to regulate gene expressionin Gram-positive bacteria:comparison of induction level and promoter strength.Appl.Environ.Microbiol.64:2763-2769).
For improving transformation efficiency, the method that recombinant plasmid expression vector is imported in the suis is preferably electrotransformation, but also can adopt method commonly used in other bioengineering field, for example heat shock method, protoplast transformation method, joint conversion method etc.
In the hyaluronic middle and later periods of fermentative Production, along with hyaluronic generation, the fermented liquid viscosity increases at present, and dissolved oxygen reduces, and has limited the growth of thalline.The present invention imports the vgb gene in the suis dexterously, by express the vgb gene in suis, can significantly improve the utilization ratio of thalline to oxygen, promotes thalli growth, makes hyaluronic output improve about 20%.The present invention is applied in the fermentation industry, can solves the disparities between supply and demand of oxygen in the high density fermentation, improve hyaluronic acid volume of production, have important industrial application value.
Description of drawings
Fig. 1 is a plasmid construction schema of the present invention.
The hyaluronic acid contents detected result of the different thalline shake flat experiments of Fig. 2.
Embodiment
Bacterial classification: intestinal bacteria E.coli JM109
The substratum that is used to make up plasmid is (g/l): peptone 10, and yeast extract 5, NaCl 10, spectinomycin 100mg/l.
Structure contain the vgb gene plasmid pEUVGB process as shown in Figure 1:
1) under the condition of the polymerase chain reaction of standard, with pBR322-vgb plasmid (Yu H M, Shi Y, ZhangY P, Yang S L, Shen Z Y.Effect of Vitreoscilla hemoglobin biosynthesis inEscherichia coli on production of poly (the and fermentativeparameters.FEMS Microbiol.Lett. of β-hydroxybutyrate), 2002,214:223-227) be template, with P
Vgb-up:GCG
AAGCTT(HindIII) AAGGAGGATGTATGGCAAAACCATAAT and P
Vgb-do:ATT
AGATCT(BglII) CTTTATTCAACCGCTTGAGC is the DNA cloning fragment that primer obtains 485bp, and its gene order is the SEQ ID NO:1 in the gene order table.
The PCR reaction conditions: 95 ℃ of sex change 1min, 57 ℃ take off fiery 1min, and 72 ℃ are extended 1min, 30 circulations.
2) with the above-mentioned DNA cloning fragment that includes the vgb gene, use restriction enzyme HindIII, after the ClaI enzyme is cut processing, be inserted into the HindIII of plasmid pEU308, between the BglII site, obtain plasmid pEUVGB.
The preparation of embodiment 2, streptococcus zooepidemicus competent cell and electric method for transformation
Bacterial classification: streptococcus zooepidemicus ATCC 39920
Used substratum is THYB (g/l): todd-hewitt broth 36.4 in the conversion, yeast extract 0.5% (w/v), spectinomycin 100mg/l.
1) preparation of competent cell
I) with streptococcus zooepidemicus overnight incubation in the THYB substratum;
II) it is inoculated in the fresh THYB substratum of 50ml inoculation back D
530Be no more than 0.05, cultivate OD
530About about 0.25 get final product;
III) add Unidasa 0.4mg/ml half an hour before cultivate finishing;
IV) 4 ℃, 8000g centrifugal 10 minutes, abandons supernatant, with 20mL 0.5M sucrose solution re-suspended cell;
V) 4 ℃, 8000g, centrifugal 10 minutes, abandon supernatant, centrifugal again with 1mL 0.5M sucrose solution re-suspended cell, abandon supernatant;
VI) cell is resuspended in the 0.5M sucrose solution that 250uL contains 10% glycerine, by using volume integral to install in the eppendorf pipe;
VII) place-80 ℃ of preservations rapidly.
2) electricity of streptococcus zooepidemicus transforms
I) in the 200uL competent cell, add 8uLDNA, ice bath 10 minutes;
II) mixed system is transferred to 2mm electricity and swashed that electricity swashs in the cup, voltage is made as 2.5kv;
III) with the ice-cold THYB of 1mL mixed system is transferred among the 10mlTHYB ice bath 30-60 minute;
IV) 37 ℃ leave standstill cultivation 1 hour;
V) 4 ℃, 6000g, centrifugal 10 minutes, thalline was resuspended and be applied to the screening of carrying out transformant on the resistant panel with 1mlTHYB, obtains the streptococcus zooepidemicus of recombinating.
Bacterial classification: reorganization streptococcus zooepidemicus
Foreign gene: vgb gene
Medium component:
1) seed culture medium
Sucrose: 20g/l, yeast powder: 10g/l, extractum carnis: 10g/l, MgSO
47H
2O:2g/l, MnSO
44H
2O:0.1g/l, KH
2PO
4: 2g/l, liquid microelement: 1ml/l, damping fluid: 40ml/l; Wherein liquid microelement contains: CaCl
2: 2g/l, MnSO
44H
2O:24mg/l, ZnCl
2: 46mg/l, CuSO
45H
2O:19mg/l.
Damping fluid contains: Na
2HPO
412H
2O:36.76g/l, NaH
2PO
412H
2O:15.98g/l, NaHCO
3: 12.5g/l.
2) fermention medium
Yeast powder: 20g/l, Na
2HPO
412H
2O:6.2g/l, MgSO
47H
2O:2g/l, K
2SO4:1.3g/l, FeSO
47H
2O:5mg/l, liquid microelement: 2.5ml/l, sucrose: 40g/l, spectinomycin: 100mg/l (the reorganization bacterium adds during fermentation).
1) cultural method
Be equipped with in the 500ml triangular flask of 50ml seed culture medium with the access of the bacterial classification on the transfering loop picking flat board, cultivated 14-16 hour on the shaking table of 200rpm, culture temperature is made as 37 ℃.By 10% inoculum size seed culture fluid is inserted in the 500ml triangular flask that fills the 50ml fermention medium then and cultivated 14 hours, culture temperature is made as 37 ℃, shaking speed 200rpm.The reorganization bacterium adds spectinomycin with the concentration of 100mg/l.
2) hyaluronic acid contents measuring method
Adopt bitter-muirShi method (Bitter T., Muri H.M.A modified uronic acid carbazolreaction.Anal.Biochem.1962,4:330-334), the concentrated sulfuric acid solution adding tool plug test tube with the 5ml0.025M sodium borate decahydrate is cooled to-20 ℃; Careful sample or the standard that adds 0.5ml, test tube seals with ground glass stopper, shakes test tube.Heating is 10 minutes in violent ebullient boiling water bath, and cool to room temperature adds 0.2ml carbazole reagent (0.125% carbazole is dissolved in the dehydrated alcohol) then, shake test tube once more, reheat is 15 minutes in boiling water bath, cool to room temperature, under 530nm, with 1cm cuvette measuring light density value, OD
530Value should be less than 0.025 to the sulfuric acid barren.Wherein standard is glucal standard acid solution 4-40ug/ml, and the preparation of the saturated deionized water of the phenylformic acid of stand-by storage can be stablized under 4 ℃ and be deposited 6 months.The measure sample preparation method of fermented liquid is as follows: a) get the 1ml fermented liquid and accurately be diluted to 4ml, 6000 left the heart 10 minutes; B) get the 1ml supernatant and join in the 4ml dehydrated alcohol, vibration, 6000 left the heart 5 minutes; C) supernatant discarded adds 2ml0.2MNaCl, place, or vibration, until final dissolving; D) add the 4ml dehydrated alcohol, vibration, 6000 left the heart 5 minutes; E) supernatant discarded, the sample that precipitation is measured as HA with the 2ml deionized water dissolving.
3) experimental result
As can be seen from Figure 2 (the 1-S.Z. streptococcus zooepidemicus, 2-changes the streptococcus zooepidemicus of empty carrier pEU308 over to, 3-changes the reorganization bacterium of recombinant plasmid pEUVGB over to), the hyaluronic acid volume of production that has the reorganization bacterium of vgb gene is higher than original bacterium.In this fermenting experiment, the hyaluronic acid volume of production of reorganization bacterium has improved about 20% with respect to original bacterium.
Sequence table
<210>1
<211>485
<212>DNA
＜213〉Vitreoscilla (Vitreoscilla)
<400>1
atgtatggca?aaaccataat?aatgaactta?aggaagaccc?tcatgttaga?ccagcaaacc 60
attaacatca?tcaaagccac?tgttcctgta?ttgaaggagc?atggcgttac?cattaccacg 120
actttttata?aaaacttgtt?tgccaaacac?cctgaagtac?gtcctttgtt?tgatatgggt 180
cgccaagaat?ctttggagca?gcctaaggct?ttggcgatga?cggtattggc?ggcagcgcaa 240
aacattgaaa?atttgccagc?tattttgcct?gcggtcaaaa?aaattgcagt?caaacattgt 300
caagcaggcg?tggcagcagc?gcattatccg?attgtcggtc?aagaattgtt?gggtgcgatt 360
aaagaagtat?tgggcgatgc?cgcaaccgat?gacattttgg?acgcgtgggg?caaggcttat 420
ggcgtgattg?cagatgtgtt?tattcaagtg?gaagcagatt?tgtacgctca?agcggttgaa 480
taaag 485
Claims (7)
1, the reorganization suis that contains the vgb gene.
2, reorganization suis according to claim 1 is characterized in that: described suis is any one in Hemolytic streptococcus, streptococcus zooepidemicus, streptococcus equi, excrement streptococcus equi, hypogalactia suis and the bacillus cholerae gallinarum.
3, according to the described reorganization of claim 2 suis, it is characterized in that: described suis is any one in A type Hemolytic streptococcus, C typothere epidemic disease suis, C type streptococcus equi, C type excrement streptococcus equi, C type hypogalactia suis and the C type bacillus cholerae gallinarum.
4, reorganization suis according to claim 2, it is characterized in that: described suis is streptococcus zooepidemicus ATCC 39920.
5, the hyaluronic method of a kind of production is to make up to contain the vgb expression carrier, and the carrier that makes up is imported in the suis, obtains the reorganization suis, and fermentation reorganization suis obtains hyaluronic acid.
6, method according to claim 5 is characterized in that: the pEUVGB that the constructed plasmid expression vector obtains for the multiple clone site that the vgb gene is inserted into carrier pEU308.
7, according to claim 5 or 6 described methods, it is characterized in that: the described method that recombinant plasmid expression vector is imported in the suis is an electrotransformation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294180B (en) * | 2008-05-26 | 2012-04-18 | 江南大学 | Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation |
| CN105368912A (en) * | 2015-10-28 | 2016-03-02 | 新疆阜丰生物科技有限公司 | Method for extracting sodium hyaluronate through spray reverse flow method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294180B (en) * | 2008-05-26 | 2012-04-18 | 江南大学 | Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation |
| CN105368912A (en) * | 2015-10-28 | 2016-03-02 | 新疆阜丰生物科技有限公司 | Method for extracting sodium hyaluronate through spray reverse flow method |
| CN105368912B (en) * | 2015-10-28 | 2018-09-14 | 新疆阜丰生物科技有限公司 | A method of spraying counter-current extracts Sodium Hyaluronate |
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