CN101037661A - Pseudoalteromonas and its usage - Google Patents
Pseudoalteromonas and its usage Download PDFInfo
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- CN101037661A CN101037661A CN 200710067313 CN200710067313A CN101037661A CN 101037661 A CN101037661 A CN 101037661A CN 200710067313 CN200710067313 CN 200710067313 CN 200710067313 A CN200710067313 A CN 200710067313A CN 101037661 A CN101037661 A CN 101037661A
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- 241000519590 Pseudoalteromonas Species 0.000 title claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 14
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000661 sodium alginate Substances 0.000 claims abstract description 12
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 12
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 235000010443 alginic acid Nutrition 0.000 claims description 33
- 229920000615 alginic acid Polymers 0.000 claims description 33
- 241000894006 Bacteria Species 0.000 claims description 20
- 241000157992 Pseudoalteromonas tetraodonis Species 0.000 claims description 18
- 238000012807 shake-flask culturing Methods 0.000 claims description 10
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- 239000012528 membrane Substances 0.000 claims description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 5
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 5
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 241000726221 Gemma Species 0.000 claims description 3
- 230000013439 flagellum movement Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 241000233866 Fungi Species 0.000 abstract 4
- 238000000855 fermentation Methods 0.000 abstract 3
- 230000004151 fermentation Effects 0.000 abstract 3
- 239000001963 growth medium Substances 0.000 abstract 3
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 abstract 3
- 238000012258 culturing Methods 0.000 abstract 2
- 241000556533 uncultured marine bacterium Species 0.000 abstract 2
- 230000004913 activation Effects 0.000 abstract 1
- 238000010612 desalination reaction Methods 0.000 abstract 1
- 239000012510 hollow fiber Substances 0.000 abstract 1
- 238000009630 liquid culture Methods 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 239000005955 Ferric phosphate Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 229940032958 ferric phosphate Drugs 0.000 description 6
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 6
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 239000007857 degradation product Substances 0.000 description 4
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
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- 239000002994 raw material Substances 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- -1 alginic acid polysaccharide Chemical class 0.000 description 1
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- 230000035784 germination Effects 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008975 immunomodulatory function Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
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- 230000036299 sexual function Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a pseudoalteromonas species and application thereof. The pseudoalteromonas species stored in NCIM, storing number is SD 227, used for producing alginate lyase. The method includes steps: 1) inoculating the pseudoalteromonas to 2216E marine bacterium solid culture medium with sodium alginate to obtain bevel fungus; 2) switching the bevel fungus to 2216E marine bacterium liquid culture medium with sodium alginate, shaking bottle and culturing to obtain liquid fungus; 3) switching the liquid fungus to triangular flask with ferment culture medium, shaking bottle and culturing to obtain fermentation liquor; 4) centrifugally removing mycobiont of the fermentation liquor, condensing to obtain alginate lyase condensing preparation. The pseudoalteromonas species is easily cultured in short generation time, fermentation liquor contains less amount and kinds of albumen, condensing desalination by using hollow fiber ultrafiltration method to obtain alginate lyase condensing preparation. The product has high activation, good stability and low cost can realize industrialized producing.
Description
Technical field
The present invention relates to a kind of Pseudoalteromonas (Pseudoalteromonas tetraodonis) and uses thereof.
Background technology
Algin claims the alginic acid polysaccharide again, is a kind of water-soluble acid polysaccharide that derives from the brown alga cell walls, mainly extracts to obtain from brown algas such as sea-tangle, bulk kelp, sargassun.China's quantity that manually cultivates kelp ranks first in the world, and accounts for 95% of world wide production.Also be the algin big producing country that is only second to the U.S. simultaneously, but the primary products that mostly are low value of producing, output about 80% for export, greatly about about 7000 to 8000 tons, remaining is 20% main as textile industry raw material.Therefore, be raw material how with resourceful low value product algin, research algin degraded product biological value and molecular mechanisms of action thereof, the relevant series product of algin that have significant application value for exploitation have and important directive function, will produce far-reaching influence to the algin industry development.
High-molecular weight algin gelation is strong, is not easy to be absorbed, and is very restricted in application facet.Through the algin of degrading in various degree, good water solubility helps absorption of human body.Studies show that, algin degradation product and derivative thereof that different molecular weight ranges, different components (M/G ratio) and different deriving methods obtain, show different physiologically actives, can be developed to multiple medical material and functional food, as anticoagulation, immunological enhancement effect, anti-tumor activity and radiation protection; The algin derivative also has reasonable AIDS resisting poison (HIV) activity
[1,2,3]The algin oligosaccharide molecular-weight average that further degraded obtains also has effects such as promotion bifidobacterium growth, broad-spectrum sterilization activity, the retentiveness that improves seed germination, promotion rudiment and root growth, enhancing fish myofibrillar protein and thermotolerance below 3KDa
[4,5,6]These have all shown the application prospect of algin degradation product as physiologic sexual function food, medicine, fodder additives, plant growth promoter.
But, present stage the research work major part for the algin degradation product also be confined in the laboratory, reason just is that algin is difficult for degraded automatically, needs suitable condition and method, it could be degraded to the oligose with different physiological roles.At present, generally adopt the dilute acid hydrolysis method to obtain the algin degradation product, the degradation speed of this method is slow, and productive rate is low, difficult quality control, the biochemical characteristic of product can not well meet the demands, and needs high temperature, high pressure, operation is complicated, the cost height, and environment polluted.By comparison, algin catenase has mild condition, efficient height, pollution-free, advantage such as Substratspezifitaet clear and definite, the biologic activity of product is strong to the DeR of algin, thereby the technology that enzyme process prepares algin oligosaccharide enjoys favor, demonstrates its tempting development prospect.
The bacterial classification that the present invention adopts be the laboratory voluntarily screening and separating obtain, through being accredited as Pseudoalteromonas (Pseudoalteromonas tetraodonis), in the preservation of Britain international standard institute of microbiology, preserving number is SD227.
Reference
1.Bland?EJ，Keshavarz?T，Bucke?C.The?influence?of?small?oligosaccharides?onthe?immune?system.Carbohydr?Res.2004，339(10)：1673-8
2.Iwamoto?M，Kurachi?M，Nakashima?T，etc.Structure-activity?relationship?ofalginate?oligosaccharides?in?the?induction?of?cytokine?production?from?RAW264.7cells.FEBS?Lett.2005，579(20)：4423-9.
3.Eun-Wha?Son，Kwang-Hee?Yang，Dong-Kwon?Rhee，etc.ImmunomodulatoryFunction?of?Murine?NK?Cell?Activity?by?Alginate.Arch?Pharm?Res.2005，28(11)：1282-6
4.Sato?R，Sawabe?T，Saeki?H.Characterization?of?Fish?Myofibrillar?Protein?byConjugation?with?Alginate?Oligosaccharide?Prepared?Using?Genetic?RecombinantAlginate?Lyase.J?Food?Sci.2005，70(1)：C58-62
5.Xiaoke?Hu，Xiaolu?Jiang，Huey-Min?Hwang，etc.Promotive?effects?ofalginate-derived?oligosaccharide?on?maize?seed?germination.Journal?of?AppliedPhycology.2004，16：73-6
6.Xiaoke?Hu，Xiaolu?Jiang，Jun?Gong，etc.Antibacterial?activity?oflyase-depolymerized?products?of?alginate.Journal?of?Applied?Phycology.2005，17(1)：57-60
7.Zobell，C.E.，1946.Marine?Microbiology.Waltham，Mass.，U.S.A，p.41-42
Summary of the invention
The purpose of this invention is to provide a kind of Pseudoalteromonas (Pseudoalteromonas tetraodonis) and uses thereof.
Pseudoalteromonas (Pseudoalteromonas tetraodonis) is in the preservation of Britain international standard institute of microbiology, preserving number is SD 227, Pseudoalteromonas (Pseudoalteromonas tetraodonis) is a Gram-negative bacteria, rod-short 1.0～2.4 μ m, amphimicrobe, by the one pole flagellar movement, no gemma, salt tolerant.
Pseudoalteromonas (Pseudoalteromonas tetraodonis) is used to produce algin catenase.
The method that is used to produce algin catenase comprises the steps:
1) with Pseudoalteromonas (Pseudoalteromonas tetraodonis) streak inoculation to the 2216E marine bacteria solid medium that contains 0.3%～0.5% sodium alginate, under 25～28 ℃ of conditions, cultivated 12～16 hours, obtain slant strains;
2) above-mentioned slant strains is forwarded in the 2216E marine bacteria liquid nutrient medium that contains 0.3%～0.5% sodium alginate, 25～28 ℃ of shake-flask culture 12～16 hours obtain liquid spawn;
3) above-mentioned 1%～3% liquid spawn is forwarded in the triangular flask of 30～50mL fermention medium, 25～28 ℃ of shake-flask culture 16～18 hours obtain fermented liquid;
4) with above-mentioned fermented liquid under 4 ℃, centrifugal 10～15 minutes of 3000rpm removes thalline, is concentrated into 5～10 times, obtains algin catenase concentrated solution preparation.
The composition of described fermention medium is unit in g/L: Secondary ammonium phosphate 11.6, sodium-chlor 32.9, ferrous sulfate 0.08, sal epsom 0.3 and dipotassium hydrogen phosphate 0.4, pH7.4～7.6.
Described concentration method is the hollow-fibre membrane hyperfiltration process.
Advantage of the present invention is, bacterial classification easily cultivates, for the time short, fermented liquid foreign protein kind is few, content is low, can obtain algin catenase concentrated solution preparation directly with the method concentrating and desalinating of tubular fibre membrane ultrafiltration.The product activity height, good stability, cost is low, can realize suitability for industrialized production.
Embodiment
Pseudoalteromonas (Pseudoalteromonas tetraodonis) is a Gram-negative bacteria, rod-short 1.0～2.4 μ m, and amphimicrobe, by the one pole flagellar movement, no gemma, salt tolerant.The pathogenic report of couple people is not arranged.Its cultural method is known, available 2216E marine bacteria substratum.Optimum growth temperature is 25～28 ℃.Pseudoalteromonas ZQ-4 can secrete the outer algin catenase of born of the same parents, and the operating temperature range of this enzyme is 30～40 ℃, and optimal reactive temperature is 40 ℃, reaction pH6～8, optimal pH 7.6; Enzyme activity is issued to 256.47U/mL shaking a bottle condition.
Embodiment 1
1) with Pseudoalteromonas (Pseudoalteromonas tetraodonis) streak inoculation to the 2216E marine bacteria solid medium that contains 0.3% sodium alginate, under 25 ℃ of conditions, cultivated 12 hours, obtain slant strains, it is unit: NaCl 30 that 2216E marine bacteria solid medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, agar 20, pH7.4～7.6
[7]
2) above-mentioned slant strains is forwarded in the 2216E marine bacteria liquid nutrient medium that contains 0.3% sodium alginate, 25 ℃ of shake-flask culture 12 hours, obtain liquid spawn, it is unit: NaCl 30 that 2216E marine bacteria liquid nutrient medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, pH7.4～7.6
[7]
3) above-mentioned 1% liquid spawn is forwarded in the triangular flask of 30mL fermention medium, 25 ℃ of shake-flask culture 16 hours, obtain fermented liquid, the composition of fermention medium is unit in g/L: Secondary ammonium phosphate 11.6, sodium-chlor 32.9, ferrous sulfate 0.08, sal epsom 0.3 and dipotassium hydrogen phosphate 0.4, pH7.4～7.6;
4) with above-mentioned fermented liquid under 4 ℃, centrifugal 15 minutes of 3000rpm removes thalline, hollow-fibre membrane is super considers and is concentrated into 5 times, obtains algin catenase concentrated solution preparation.
Embodiment 2
1) with Pseudoalteromonas (Pseudoalteromonas tetraodonis) streak inoculation to the 2216E marine bacteria solid medium that contains 0.5% sodium alginate, under 28 ℃ of conditions, cultivated 16 hours, obtain slant strains, it is unit: NaCl 30 that 2216E marine bacteria solid medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, agar 20, pH7.4～7.6;
2) above-mentioned slant strains is forwarded in the 2216E marine bacteria liquid nutrient medium that contains 0.5% sodium alginate, 28 ℃ of shake-flask culture 16 hours, obtain liquid spawn, it is unit: NaCl 30 that 2216E marine bacteria liquid nutrient medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, pH7.4～7.6;
3) above-mentioned 3% liquid spawn is forwarded in the triangular flask of 50mL fermention medium, 28 ℃ of shake-flask culture 18 hours, obtain fermented liquid, the composition of fermention medium is unit in g/L: Secondary ammonium phosphate 11.6, sodium-chlor 32.9, ferrous sulfate 0.08, sal epsom 0.3 and dipotassium hydrogen phosphate 0.4, pH7.4～7.6;
4) with above-mentioned fermented liquid under 4 ℃, centrifugal 15 minutes of 3000rpm removes thalline, hollow-fibre membrane is super considers and is concentrated into 10 times, obtains algin catenase concentrated solution preparation.
Embodiment 3
1) with Pseudoalteromonas (Pseudoalteromonas tetraodonis) streak inoculation to the 2216E marine bacteria solid medium that contains 0.4% sodium alginate, under 27 ℃ of conditions, cultivated 14 hours, obtain slant strains, it is unit: NaCl 30 that 2216E marine bacteria solid medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, agar 20, pH7.4～7.6;
2) above-mentioned slant strains is forwarded in the 2216E marine bacteria liquid nutrient medium that contains 0.4% sodium alginate, 27 ℃ of shake-flask culture 14 hours, obtain liquid spawn, it is unit: NaCl 30 that 2216E marine bacteria liquid nutrient medium is formed in g/L, peptone 5, yeast extract 1, high ferric phosphate 0.01, pH7.4～7.6;
3) above-mentioned 2% liquid spawn is forwarded in the triangular flask of 40mL fermention medium, 27 ℃ of shake-flask culture 17 hours, obtain fermented liquid, the composition of fermention medium is unit in g/L: Secondary ammonium phosphate 11.6, sodium-chlor 32.9, ferrous sulfate 0.08, sal epsom 0.3 and dipotassium hydrogen phosphate 0.4, pH7.4～7.6;
4) with above-mentioned fermented liquid under 4 ℃, centrifugal 15 minutes of 3000rpm removes thalline, hollow-fibre membrane is super considers and is concentrated into 8 times, obtains algin catenase concentrated solution preparation.
Claims (5)
1. a Pseudoalteromonas (Pseudoalteromonas tetraodonis), it is characterized in that, in the preservation of Britain international standard institute of microbiology, preserving number is SD 227, and Pseudoalteromonas (Pseudoalteromonas tetraodonis) is a Gram-negative bacteria, rod-short 1.0～2.4 μ m, amphimicrobe, by the one pole flagellar movement, no gemma, salt tolerant.
2. the purposes of Pseudoalteromonas (Pseudoalteromonas tetraodonis) according to claim 1 is characterized in that it is used to produce algin catenase.
3. the purposes of a kind of Pseudoalteromonas according to claim 2 (Pseudoalteromonastetraodonis) is characterized in that the described method that is used to produce algin catenase, comprises the steps:
1) with Pseudoalteromonas (Pseudoalteromonas tetraodonis) streak inoculation to the 2216E marine bacteria solid medium that contains 0.3%～0.5% sodium alginate, under 25～28 ℃ of conditions, cultivated 12～16 hours, obtain slant strains;
2) above-mentioned slant strains is forwarded in the 2216E marine bacteria liquid nutrient medium that contains 0.3%～0.5% sodium alginate, 25～28 ℃ of shake-flask culture 12～16 hours obtain liquid spawn;
3) above-mentioned 1%～3% liquid spawn is forwarded in the triangular flask of 30～50mL fermention medium, 25～28 ℃ of shake-flask culture 16～18 hours obtain fermented liquid;
4) with above-mentioned fermented liquid under 4 ℃, centrifugal 10～15 minutes of 3000rpm removes thalline, is concentrated into 5～10 times, obtains algin catenase concentrated solution preparation.
4. the purposes of a kind of Pseudoalteromonas according to claim 3 (Pseudoalteromonastetraodonis), the composition that it is characterized in that described fermention medium is unit in g/L: Secondary ammonium phosphate 11.6, sodium-chlor 32.9, ferrous sulfate 0.08, sal epsom 0.3 and dipotassium hydrogen phosphate 0.4, pH7.4～7.6.
5. the purposes of a kind of Pseudoalteromonas according to claim 3 (Pseudoalteromonastetraodonis) is characterized in that described concentration method is the tubular fibre membrane method.
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| CN102168039A (en) * | 2010-12-10 | 2011-08-31 | 厦门大学 | Method for screening extracellular algae-killing protein marine bacteria |
| CN102311930A (en) * | 2010-07-02 | 2012-01-11 | 中国海洋大学 | Alginate lyase produced by pseudomonas.sp.HZJ216 |
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| CN103160451A (en) * | 2013-01-21 | 2013-06-19 | 浙江省舟山市水产研究所 | Pseudoalteromonas and purpose thereof |
| CN103695341A (en) * | 2013-12-16 | 2014-04-02 | 江南大学 | Alginate lyase secreted from marine bacterium and preparation method thereof |
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