CN103834593B - A kind of secondary coccus and application thereof - Google Patents
A kind of secondary coccus and application thereof Download PDFInfo
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- CN103834593B CN103834593B CN201410077382.8A CN201410077382A CN103834593B CN 103834593 B CN103834593 B CN 103834593B CN 201410077382 A CN201410077382 A CN 201410077382A CN 103834593 B CN103834593 B CN 103834593B
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Abstract
The object of this invention is to provide a kind of secondary coccus, China Committee for Culture Collection of Microorganisms's common micro-organisms center of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences within 01 month 10 days, has been preserved in 2014, does is deposit number CGMCC? No.8712.The present invention screening bacterium for the production of fucosan sulfatase, chondroitinase and hyaluronic acid glucosamine thuja acid enzyme, can degrade efficiently fucoidan, chondroitin sulfate and hyaluronic acid.
Description
Technical field
The invention belongs to marine microorganism triage techniques field, be specifically related to a kind of secondary coccus and application thereof, namely a strain has bacterium and the application thereof of fucosan sulfatase, chondroitinase and hyaluronic acid glucosamine thuja acid enzyme throughput.
Background technology
Fucoidan (Fucoidan) is a kind of sulfuric acid mixed polysaccharide of high molecular, primarily of L-fucose and sulfate group composition, also containing semi-lactosi, glucose, wood sugar, rhamnosyl, seminose, uronic acid etc., be mainly derived from brown alga and echinoderm.Research finds that it has multiple physiologically active, and comprise anticoagulation, antithrombotic, raising immunizing power, anti-oxidant etc., therefore Fucoidan becomes the main attack emphasis of current natural marine active substance.But because the molecular weight of Fucoidan is huge, have from several ten thousand to hundreds of thousands of, had a strong impact on its assimilated efficiency, simultaneously Fucoidan also has certain viscosity, also greatly hinder its application as medicine.The Fucoidan of high molecular is degraded to low-molecular-weight carbohydrate, can solves an above-mentioned application difficult problem, be also the important means of carrying out the research of Fucoidan structure effect simultaneously.The best approach of degrading to Fucoidan is at present enzymolysis process, and the method specificity is high, and process easily controls, and object product assay is high, by force functional.This enzyme is mainly derived from microbes producing cellulase, and domestic and international at present have several sections of reports to this type of microbes producing cellulase, but also do not have commercial zymin to occur, mainly the enzymatic productivity of microorganism not yet reaches the requirement of production.Therefore, the superior strain finding enzyme is the important channel expanding Fucoidan application at present.
Chondroitin sulfate (ChondroitionSulfate, CS) be a kind of acidic mucopolysaccharide---glycosaminoglycan, the biomacromolecule that the dissacharide units mainly formed by glucuronic acid and hexosamine is alternately formed by connecting, the number of dissacharide units is usually at about 50-70, and relative molecular weight is greatly between 5000-50000Da.This polysaccharide content in mammiferous reticular tissue is more, as cartilage, ligament, animal larynx bone, nasal bone, sarolemma, tracheae and vessel wall etc.Different according to the position of sulfate in structure, chondroitin sulfate can be divided into the multiple isomer such as A, B, C, D, E, F, H, at present as medicinal mainly A and C isomer.If the sulfate in molecule on semi-lactosi is on 4, claim chondroitin sulfate A (CSA) or chondroitin-4-suleate; If the sulfate in molecule on semi-lactosi is on 6, be then called chondroitin sulfate C or 6-chondroitin sulfate, the chondroitin sulfate of general indication is the mixture of A and C isomer.Chondroitin sulfate has stronger reducing blood lipid and buffering blood coagulation resisting function, is mainly used in clinically preventing coronary heart disease and atherosclerosis.New research finds to show, when the molecular weight of chondroitin sulfate is reduced to 2-10kDa, its drug effect is more obvious, and have better curative effect to atherosclerosis, rheumatic inflammation and wound healing, therefore preparing low-molecular-weight chondroitin sulfate is key.The CS of high molecular is degraded to low molecular weight product is illustrate the most effective way of its structure, nature and function relation simultaneously.The preparation of current low-molecular weight chondroitin sulfate often adopts acid-hydrolysis method, ion exchange method and enzymolysis process.First two method needs more complicated equipment and operation and can cause environmental pollution, the mild condition that enzymolysis process needs and easily controlling, and is optimal one in aforesaid method.Chondroitinase can glycosaminoglycan specifically in degradation of cell epimatrix composition as hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin etc., degraded product is unsaturated disaccharide and oligosaccharides.The current main chondroitinase that extracts from microorganism is abroad to obtain highly purified zymin, the bacterial strain of this enzyme of product reported mainly contains proteus vulgaris (Proteusvulgaris), Flavobacterium heparinum (Flavobacteriumheparinum) and Proteus mirabilis (Proteusmirabilis) etc.
Unidasa (Hyaluronidase) is to catalyzed degradation hyaluronic acid (Hyaluronicacid, the common name of enzyme HA), according to the difference of hyaluronic acid enzyme mechanism, they are divided into 3 classes: (2) glucosaminidase (EC3.2.1.35), acting on β-Isosorbide-5-Nitrae glycosidic link, is a kind of lytic enzyme, end product is mainly tetrose, also can act on chrondroitin or chondroitin sulfate; (2) glucosamine thuja acid enzyme (EC3.2.1.36), acts on β-1,3 glycosidic link, and be also lytic enzyme, main degradation products is tetrose, selective degradation hyaluronic acid.(3) hyaluronate lyase (EC4.2.2.1), acts on β-Isosorbide-5-Nitrae glycosidic link, obtains 4 by β-cancellation mechanism, 5 unsaturated disaccharide.Recently for over ten years, people show keen interest to HA and Unidasa, research finds that Unidasa is by the HA in degraded tissue, increase the permeability of film, promote the diffusion of injection liquid, therefore it can be applied to therapeutic process, to promote that injection liquid absorbs, improve local anaesthesia effect, and prevent destruction that is subcutaneous and muscle tissue.In fundamental research, Unidasa is also good toolenzyme, can be used for the research of polysaccharide and the preparation of oligosaccharide.Increasing evidence shows, the biological activity of the HA that relative molecular mass is low is significantly different from HA, and a lot of researchist starts to carry out the research that enzymic degradation HA prepares oligosaccharides in recent years, thus can inquire into its biological significance further.And microorganism obtains Unidasa best source, the gram negative bacterium with this enzyme throughput reported at present mainly contains Aeromonas (Aeromonas), bacterioide (Bacteroides), clostridium (Fusobacterium) and vibrios (Vibrio) etc.
Summary of the invention
The object of this invention is to provide a kind of secondary coccus and application thereof, i.e. a kind of bacterium simultaneously with fucosan sulfatase, chondroitinase and hyaluronic acid glucosamine thuja acid enzyme throughput and the enzyme produced thereof, can effectively to degrade fucoidan, chondroitin sulfate and hyaluronic acid, for the application and the research of structure effect better realizing three kinds of polysaccharide provides effective ways.
Secondary coccus (paracoccussp.) MD2 of the present invention, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences for 01 month 10 days in 2014, deposit number is CGMCCNo.8712.
The bacterium of the present invention's screening is for the production of fucosan sulfatase, chondroitinase and hyaluronic acid glucosamine thuja acid enzyme;
The bacterium of the present invention's screening is for fucoidan of degrading, chondroitin sulfate and hyaluronic acid.
The viable bacteria of paracoccus bacterium MD2 of the present invention is for the preparation of bacterium liquid; Obtained bacterium liquid may be used for extracting intracellular enzyme, thus for fucoidan of degrading, chondroitin sulfate and hyaluronic acid.
Accompanying drawing explanation
The electrophoresis result figure of Fig. 1: cellular enzymatic cleavage CSA different time, wherein: S, use chondroitin sulfate A (CSA) BC enzyme liberating CSA react the product of 24h; E, intracellular enzyme; CSA, chondroitin sulfate; 3h, 5h, 7h, 10h, 24h, react 3h respectively with cellular enzymatic cleavage CSA, the product of 5h, 7h, 10h, 24h.
Fig. 2: the High Performance Liquid Chromatography/Mass Spectrometry analyzing total ionic current spectrogram of chondroitin sulfate A (CSA) enzymolysis product.
Fig. 3: the High Performance Liquid Chromatography/Mass Spectrometry analyzing total ionic current spectrogram of hyaluronic acid enzymolysis product.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
One, the screening of secondary coccus
Microorganism enrichment, primary dcreening operation, multiple sieve are carried out to the seawater sample in Mai Dao marine site, Qingdao City, screening zymogenic bacteria.Liquid culture is carried out to zymogenic bacteria, collects microbial cells and extract intracellular enzyme.
Concrete technical scheme is as follows:
(1) microorganism enrichment culture: get seawater sample 2.5mL under aseptic condition and access 50mL enrichment medium (Fucoidan0.2%, peptone 1%, ammonium nitrate 1.6mg/mL, Sodium phosphate dibasic 8mg/mL, with crossing the preparation of film seawater, pH nature) cultivate, at 25 DEG C, the constant-temperature shaking incubator of 150rpm cultivates 5 days.After cultivation terminates, get above-mentioned enrichment culture liquid 2.5mL and access enrichment medium repetition enrichment twice.
(2) microbes producing cellulase primary dcreening operation: after being diluted by enrichment culture liquid, coats selective medium (ammonium nitrate 5mg/mL, agar 2%, used film seawater prepare, pH nature for Fucoidan0.2%, peptone 0.2%) on flat board, 25 DEG C of cultivations 5 days.By single bacterium colonies different for forms all in flat board, carry out purifying cultivation, then access slant medium (2216E agar, with distilled water preparation, pH nature), be cultured in 25 DEG C and cover with inclined-plane and be placed in 4 DEG C of Refrigerator stores.
(3) microbes producing cellulase sieves again: above-mentioned slant strains is seeded to respectively 15mL liquid nutrient medium (Fucoidan0.2%, peptone 1%, ammonium nitrate 5mg/mL, with crossing the preparation of film seawater, pH nature), in 25 DEG C, the shaking table of 180r/min is cultivated, and measures polysaccharide content with methyne blue laws.Result obtains a strain bacterium MD2, cultivates polysaccharide content decline after 72 hours and reaches 51%.
(4) form of zymogenic bacteria MD2, Physiology and biochemistry and molecular biology identification: by zymogenic bacteria streak inoculation in 2216E agar plate, observation by light microscope ne ar and Physiology and biochemistry identify colony characteristics to be bacterium colony are white, rounded protuberance shape, opaque, surface wettability, neat in edge, cell Gram-negative, the molecular biology identification that 16SrDNA carries out belongs to paracoccus (Paracoccussp.).Bacterial strain is on 2216E agar plate after 25 DEG C of cultivation 24h, and bacterium colony is white, and rounded protuberance shape is opaque, surface wettability, neat in edge, Gram-negative.Coated by this bacterium on 2216E agar plate, namely 25 DEG C of cultivations grow single bacterium colony in 24-48 hour.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences for 01 month 10 days in 2014, deposit number is CGMCCNo.8712.
Two, the preparation of secondary coccus bacterium liquid
Be that the secondary coccus MD2 of CGMCCNo.8712 is inoculated in liquid nutrient medium (Fucoidan0.2% by deposit number, peptone 1%, ammonium nitrate 5mg/mL, with crossing the preparation of film seawater, pH nature) in, 25 DEG C, cultivate 12h under 180rpm condition, obtain seed culture fluid, seed culture fluid is inoculated in fresh liquid nutrient medium by 5% inoculum size, in 25 DEG C, cultivate 72h under 150rpm condition, obtain secondary coccus bacterium liquid.
Three, the preparation of secondary coccus intracellular enzyme
By the bacterium liquid of secondary coccus MD2 in 4 DEG C, the centrifugal 5-10 minute of 10000 × g; abandon supernatant; get precipitation; precipitate with the Tris-HCl solubilize of a small amount of 20mM, pH8.0, under ice bath protection, 500w condition carries out ultrasonic disruption cell 15min; in 4 DEG C of centrifugal 10min; abandon precipitation, supernatant is intracellular enzyme, 4 DEG C of preservations.
Four, the detection of secondary coccus enzymatic productivity and degradation of polysaccharide ability
1, secondary coccus produces the ability detection of enzyme and degraded fucoidan
Embodiment 1:
Get intracellular enzyme to dissolve with the Tris-HCl of Fucoidan(pH8.0,20mM of 0.2%) equal-volume mixes, and in 25 DEG C, 120rpm shaking table shaking culture 2 hours, measures reducing sugar content with Tripotassium iron hexacyanide method.1 enzyme activity unit is defined as 25 DEG C, under pH8.0 condition, the enzyme amount that 1 nmole reducing sugar of generation per hour needs.After measured, produce enzyme activity after secondary coccus cultivation 72h and reach 150U/mL.
Embodiment 2:
(Fucoidan0.2%, ammonium nitrate 0.2% used film seawater to prepare zymogenic bacteria MD2 to be inoculated in liquid nutrient medium, pH nature), 25 DEG C, cultivate 72h under 150rpm condition, every 24h aseptically samples 1mL, at 80 DEG C of enzyme 15min that go out, get supernatant liquor in the centrifugal 10min of 10000rpm.With high performance size exclusion chromatography, (chromatographic condition is as follows: Aglient1100 high performance liquid chromatograph; TSK-gelG3000PW × 1(30cm × 7.5mm) chromatographic column; 40 DEG C of column temperatures; 0.2MNaCl is moving phase; 0.5mL/min flow velocity; Composition distribution) detect the change of polysaccharide molecular weight.After result shows to cultivate 0h, 24h, 48h, 72h, retention time is that the Fucoidan content of 13.6min is respectively 100%, 78%, 45%, 30%.Prove that the bacterial strain of the present invention's screening has the performance of well degraded fucoidan.
2, secondary coccus produces the ability detection of enzyme and degraded chondroitin sulfate
Embodiment 3:
Get 1mL intracellular enzyme to mix with chondroitin sulfate A (CSA) (CSA) (dissolving with the Tris-HCl of pH8.0,20mM) equal-volume of 1mL0.2%, in 37 DEG C of reaction 60min, then through 100 DEG C of water-bath 5min termination reactions.In the uv-absorbing of 232nm working sample.Blank for reacting again after the enzyme that first goes out.Under 1 enzyme activity unit is defined as 37 DEG C of conditions, per minute catalysis generates the enzyme amount that 1 micromole's disaccharide needs.After measured, produce intracellular enzyme vigor after secondary coccus cultivation 72h and reach 2.1U/mL.
Embodiment 4:
Get the Tris-HCl preparation of the CSA(20mMpH8.0 of 100 μ L20mg/mL) add in 2mL centrifuge tube, then add intracellular enzyme 200 μ L, mixing, be placed in 28 DEG C, the constant temperature oscillator of 110r/min, reaction 24h.When 3h, 5h, 7h, 10h, 24h are carried out in reaction, take out 20 μ L respectively, go out enzyme 10min in 100 DEG C of loft drier, then in the centrifugal 5min of 10000r/min, gets supernatant liquor.Carry out Polyacrylamide Gel Electrophoresis to above-mentioned sample, condition is as follows: concentrated gum concentration 5%, resolving gel concentration 15%, carries out electrophoresis at room temperature constant voltage 200V and be about 30min.Electrophoresis terminates rear use 0.5% A Li Xinlan (being dissolved in 2% acetic acid solution) and carries out dyeing 30min, then decolours with 2% acetic acid solution.Electrophoresis result is shown in accompanying drawing 1, as seen from the figure enzymolysis 3,5,7,10,24h all there will be oligose fragment, along with the prolongation of enzymolysis time, CSA content reduces gradually, and oligosaccharide content increases gradually.Show that the intracellular enzyme that bacterial strain MD2 produces has significant degradation effect to CSA.
Embodiment 5:
Get the Tris-HCl preparation of the CSA(20mMpH8.0 of 100 μ L20mg/mL) add in 2mL centrifuge tube, then add intracellular enzyme 200 μ L, mixing.Be placed in 28 DEG C, the constant temperature oscillator of 110r/min, reaction 24h.When 3h, 5h, 7h, 10h, 24h are carried out in reaction, take out 20 μ L respectively, go out enzyme 10min in 100 DEG C of loft drier, then in the centrifugal 5min of 10000r/min, gets supernatant liquor.Adopt ion pairing HPLC-MS enzyme analysis hydrolysis products, condition is as follows: high performance liquid chromatograph (Agilent1100capillaryLC), chromatographic column (AgilentZORBAXSB-C180.5mm × 250mm, 5 μm).Mobile phase A: the 15mM n-amylamine aqueous solution, pH7.0; Mobile phase B: the acetonitrile solution of 75%, containing 15mM n-amylamine, pH7.0.Sample concentration 10 μ g/ μ L(mobile phase A is dissolved), applied sample amount 2 μ L.First use mobile phase A wash-out 5min, then use Mobile phase B (16 – 56%) to carry out linear gradient elution 65min.Enzymolysis product is separated through ion pairing high performance liquid chromatography, then enters mass spectrum analysis, and compartment analysis the results are shown in accompanying drawing 2, subordinate list 1.Complicated chondroitin sulfate A (CSA) enzymolysis product is well separated on ion pairing reverse-phase chromatographic column, mass spectrometry results shows, the enzymolysis product mainly polymerization degree is the unsaturated even number sugar of 2-22, basic structure is the glucuronic acid that Δ AN (AN) n(Δ A represents containing unsaturated double-bond, and A represents glucuronic acid; N represents acetylamino galactosamine, n=0-10), containing a unsaturated double-bond, be made up of the repetition disaccharide unit of glucuronic acid and acetylamino galactosamine, each monose have 1 sulfate radical.Illustrate that the intracellular enzyme that this bacterial strain MD2 produces is chondroitinase, can the β-1 of cracking chondroitin sulfate, 4 glycosidic links, between the carbon 4 and 5, carbon of the uronic acid of non-reducing end, dehydration defines a unsaturated double-bond, thus generates the even number sugar of the repetition disaccharides composition of glucuronic acid and acetylamino galactosamine.
Table 1: the mass spectrometry results of chondroitin sulfate A (CSA) enzymolysis product
Note: M represents sugar, and PTA represents n-amylamine; Between Δ A carbon 4 and 5, carbon, dehydration defines the glucuronic acid of a unsaturated double-bond; A represents glucuronic acid; N represents acetylamino galactosamine; S represents sulfate radical
3, secondary coccus produces enzyme and hyaluronic ability of degrading detection
Embodiment 6:
The hyaluronic acid (preparing with the Tris-HCl of 20mMpH8.0) getting 100 μ L20mg/mL adds in 2mL centrifuge tube, then adds intracellular enzyme 200 μ L, mixing.Be placed in 28 DEG C, the constant temperature oscillator of 110r/min, reaction 24h.When 3h, 5h, 7h, 10h, 24h are carried out in reaction, take out 20 μ L respectively, go out enzyme 10min in 100 DEG C of loft drier, then in the centrifugal 5min of 10000r/min, gets supernatant liquor.Adopt HPLC-MS enzyme analysis hydrolysis products structure, condition is as follows: high performance liquid chromatograph (Agilent1100), chromatographic column (2.0 × 150mm, 200A, Phenomenex, Torrance, CA).Mobile phase A: 5mM ammonium acetate aqueous solution; Mobile phase B: the acetonitrile solution of 98%, containing 5mM ammonium acetate.Sample concentration 10 μ g/ μ L(50% acetonitrile solution dissolves), applied sample amount 4 μ L.First use 2% mobile phase A wash-out 5min, then use mobile phase A (2-30%) to carry out linear gradient elution 90min.Enzymolysis product is separated through high performance liquid chromatography, then enters mass spectrum analysis.Compartment analysis the results are shown in accompanying drawing 3, subordinate list 2.Hyaluronic acid enzymolysis product is well separated on hydrophilic chromatographic post.Mass spectrometry results shows, in enzymolysis product, mainly the polymerization degree is the even number sugar of 2-16, and basic structure is Δ AN (AN)
n(Δ A represents the glucuronic acid containing unsaturated double-bond; A represents glucuronic acid, and N represents acetylglucosamine), containing a unsaturated double-bond, be made up of the repetition disaccharide unit of glucuronic acid and acetylglucosamine.Illustrate in this intracellular enzyme containing hyaluronate lyase (EC4.2.2.1), can the hyaluronic β-1 of enzymolysis, 4 glycosidic links, generate the even number polysaccharide be made up of the repetition disaccharides of glucuronic acid and acetylglucosamine, on the uronic acid of non-reducing end, between carbon 4 and carbon 5, dehydration defines a unsaturated double-bond.Meanwhile, mass spectrometry results shows also containing the unsaturated sugar of hyaluronic acid odd number that structural unit is Δ A (NA) in enzymolysis product, containing a unsaturated double-bond, is glucuronic acid at reducing end.This intracellular enzyme is described also containing hyaluronic acid glucosamine thuja acid enzyme (EC3.2.1.36), can β-1,3 glycosidic link in specific for hydrolysis hyaluronic acid between glucuronic acid and acetylglucosamine, generate a saturated glucuronic acid at reducing end.
Table 2: the mass spectrometry results of hyaluronic acid enzymolysis product
Note: Δ A represents dehydration between carbon 4 and carbon 5 and defines the glucuronic acid of a unsaturated double-bond; A represents glucuronic acid, and N represents acetylglucosamine
Embodiment 7:
The intracellular enzyme 1mL produced by MD2 mixes with the fucoidan of 1mL2%, chondroitin sulfate, hyaluronic acid (all dissolving with the Tris-HCl of pH8.0,20mM) respectively, after 37 DEG C of reaction 10h, utilize the reducing sugar content of Tripotassium iron hexacyanide method respectively initial and end time of assaying reaction system immediately.Result shows, the reducing sugar content of fucoidan system, from 1.58 μm of ol/mL, rises to 2.56 μm of ol/mL; The reducing sugar content of chondroitin sulfate system rises to 15.62 μm of ol/mL from 1.35 μm of ol/mL; The reducing sugar content of hyaluronic acid system rises to 26.45 μm of ol/mL from 1.44 μm of ol/mL.
Above-mentioned experimental result shows, the present invention screens the zymogenic bacteria MD2 obtained and has degraded fucoidan, chondroitin sulfate and hyaluronic ability simultaneously, has good application prospect.
Claims (5)
1. a secondary coccus, it is characterized in that, described secondary coccus is secondary coccus (paracoccussp.) MD2, within 01 month 10 days, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences in 2014, deposit number is CGMCCNo.8712.
2. the application of secondary coccus according to claim 1 in production fucosan sulfatase, chondroitinase and hyaluronic acid glucosamine thuja acid enzyme.
3. the application of secondary coccus according to claim 1 in degraded fucoidan.
4. the application of secondary coccus according to claim 1 in degraded chondroitin sulfate.
5. the application of secondary coccus according to claim 1 in degraded hyaluronic acid.
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| CN1206344C (en) * | 2003-07-28 | 2005-06-15 | 中国海洋大学 | Method for producing fucan sulfatase by means of bacteria |
| KR20080059850A (en) * | 2006-12-26 | 2008-07-01 | 주식회사 글루칸 | A novel micoorganism of pseudoalteromonas sp. and method for producing oligofucoidan using the same and produts thereof |
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| CN102220270B (en) * | 2011-05-18 | 2013-05-15 | 江南大学 | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate |
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