CN1544615A - Vibrio and process for producing guluronicacid lyase by using the same - Google Patents
Vibrio and process for producing guluronicacid lyase by using the same Download PDFInfo
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- CN1544615A CN1544615A CNA2003101057278A CN200310105727A CN1544615A CN 1544615 A CN1544615 A CN 1544615A CN A2003101057278 A CNA2003101057278 A CN A2003101057278A CN 200310105727 A CN200310105727 A CN 200310105727A CN 1544615 A CN1544615 A CN 1544615A
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- vibrios
- lyase
- vibro
- acid lyase
- guluronic acid
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Abstract
The invention is a vibrion, its characteristic: it is Vibro sp.WYB, conservation unit is Chinese Typical Culture Conversation Center, and conservation number is M203075. When preparing alpha-1, 4-L-gulonic aldehydic acid lyase by using it, inoculate the vibrion in a liquid culture medium containing algin, shaking bottle and cultivating at constant temperature 20-30 deg.C, centrifugate and eliminate thalli, deposit the supernatant by ammonium sulphate at saturation 65-85%, and separate the deposit by ion exchange chromatography to obtain pure gulonic aldehydic acid lyase, which is stable at 20-45deg.C, the optimum acting temperature is 35 deg.C and the optimum pH value is 7. The strains is stable, the fermenting time is short, the yield of gulonic aldehydic acid lyase is high and the lyase is easy to separate and has good stability, strong specificity, and high activity and can act as tool enzyme of analyzing brown alga glycocoll chain.
Description
Technical field
The present invention relates to a kind of vibrios and produce α-1, the method for 4-L-guluronic acid lyase with it.
Background technology
Algin is the main component of brown alga cell walls, the linear molecule of being made up of mannuronic acid and guluronic acid.It is relevant to contain guluronic acid in many biological functions of algin and its molecule.It is clear and definite that the guluronic acid oligosaccharides has structure, stable performance, and characteristics such as easy absorption are raw material with the algin, adopt acid system, oxidation style and enzyme process etc. all can obtain the guluronic acid oligosaccharides, but the oligosaccharide structure difference that different methods obtains.Enzyme process prepares the characteristics that oligosaccharides has mild condition, controls easily, is the emphasis of research and development.Vibrios can be used for preparing alginate lyase, as in CN1445365A, disclosing a kind of method for preparing alginate lyase with vibrios (Vibro SP QY101), a kind of method for preparing alginate lyase with Vibrio flurialis (Vibro fluvialis) is disclosed in CN1333336A, but the alginate lyase that is produced by these two kinds of vibrios lacks unicity, this is guluronic acid and mannuronic acid in the algin because these vibrios can be degraded simultaneously, in the algin of can not degrading due to the guluronic acid singlely.
The purpose of this invention is to provide a kind of vibrios Vibro sp.WYB, this vibrios can single generation guluronic acid lyase, and α-1 in the single-minded degraded algin, and the 4-L-guluronic acid can overcome the above-mentioned deficiency of prior art.
Summary of the invention
A kind of vibrios is characterized in that this vibrios is Vibro sp.WYB; Depositary institution is Chinese typical culture collection center, and preserving number is M203075.
Vibrios Vibro SP WYB is used to produce α-1,4-L-guluronic acid lyase.
A kind of method of producing the guluronic acid lyase with vibrios Vibro sp.WYB, comprise vibrios is inoculated in the liquid nutrient medium that contains algin, the constant temperature shake-flask culture, centrifugal removal thalline, supernatant liquor obtains thick guluronic acid lyase with ammonium sulfate precipitation, and this crude enzyme liquid further separates with ion-exchange chromatography.
Vibrios Vibro sp.WYB of the present invention stable performance, the guluronic acid lyase kind of producing with it is single, and specificity is strong, the vigor height, because the α-1 in its single-minded hydrolysis of brown algae glue, 4-L-guluronic acid glycosidic link, and at unsaturated double-bond of non-reducing end generation.This vibrios can be continuously fermented, and thick guluronic acid lyase liquid only needs just can obtain pure guluronic acid lyase through a chromatographic separation and purification, and the vigor of this enzyme is unaffected.
Description of drawings and embodiment
Accompanying drawing 1 is a vibrios strain aspect graph of the present invention.
Accompanying drawing 2 is vibrios 16s rRNA gene sequencing figure of the present invention.
As can be seen from Figure 1, vibrios Vibro sp.WYB end of the present invention is given birth to single flagellum, and the length of flagellum and thalline are long almost equal.According to 16
sRRNA gene sequencing and biochemical indicator analysis thereof confirm that this bacterium is the novel species of Vibrio.The seed culture medium of this vibrios is formed: algin 0.2% (weight percentage, down together)-0.5%; Sodium-chlor 1-3%; Yeast extract 0.1-0.3%; Peptone 0.5-1%, pH7-7.5, leavening temperature 25-30 ℃, obtain Vibro sp.WYB vibrios, its depositary institution is Chinese typical culture collection center, is called for short CCTCC, preserving number is M203075.During preparation guluronic acid lyase, vibrios (is contained algin 0.5-1.0% in liquid nutrient medium, 2-3%NaCl, the 0.5-1.0% yeast extract) cultivates in, at 25 ℃, the 150rpm bottom fermentation was cultivated 12-48 hour, the fermented liquid high speed centrifugation is collected supernatant liquor, in supernatant liquor, add ammonium sulfate, make its saturation ratio 50-55%, centrifugal removal precipitation, collect supernatant liquor, continue to add ammonium sulfate, make saturation ratio reach 65-85% to clear liquid, centrifugal collecting precipitation obtains guluronic acid lyase crude enzyme liquid after the dialysis.The crude enzyme liquid of gained is separated with DEAE-A25 (XK26/20) post, with containing sodium phosphate (10mmol/L, pH7) sodium chloride solution (0-1mol/L) gradient elution, collect active ingredient, adopt press filtration to concentrate after washing thoroughly, lyophilize obtains pure enzyme, 35 ℃ of this enzyme optimum tempss, best pH7.0, molecular weight 41.3KD.DEAE-A25 in the present embodiment can use DEAE-Sephacel or Q-Sepharose F.F instead.
Respectively algin, guluronic acid and mannuronic acid are made into 0.02% aqueous solution with 10mmol/L phosphoric acid salt (PH7), in 35 ℃ of thermostatical color comparison ponds, add 20 μ L guluronic acid lyases (68u/mg), utilization enzyme kinetics dedicated analysis software dynamic measurement ultraviolet (235nm) absorption value on UV-2 102PCS uv analyzer, along with time lengthening, absorption value significantly increases in guluronic acid solution and the alginate solution, and mannuronic acid solution absorption value is constant substantially, shows that this enzyme has single-minded degraded guluronic acid function.
Claims (5)
1 one kinds of vibrios is characterized in that this vibrios is Vibro sp.WYB, and depositary institution is CCTCC, and preserving number is M203075.
2 vibrios Vibro sp.WYB are used to produce α-1,4-L-guluronic acid lyase.
3 one kinds of methods of producing the guluronic acid lyase with vibrios Vibro sp.WYB, comprise vibrios is inoculated in the liquid nutrient medium that contains algin, the constant temperature shake-flask culture, centrifugal removal thalline, supernatant liquor obtains thick guluronic acid lyase with ammonium sulfate precipitation, and this crude enzyme liquid further separates with ion-exchange chromatography.
4 methods as claimed in claim 3, it is characterized in that described thermostat temperature is 20-30 ℃, shaking bottle speed is 100-300rpm, incubation time is 12-48 hour, the saturation ratio of described ammonium sulfate is 65-85%, and the filler of described used in chromatograph is DEAE-A25, DEAE-Sephacel or Q-Sepharose F.F.
5 methods as claimed in claim 3 is characterized in that described liquid nutrient medium contains the 0.5-1.0% algin, 2-3%NaCI, and 0.5-1.0% yeast extract.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101057278A CN1544615A (en) | 2003-11-26 | 2003-11-26 | Vibrio and process for producing guluronicacid lyase by using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101057278A CN1544615A (en) | 2003-11-26 | 2003-11-26 | Vibrio and process for producing guluronicacid lyase by using the same |
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| CN1544615A true CN1544615A (en) | 2004-11-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2003101057278A Pending CN1544615A (en) | 2003-11-26 | 2003-11-26 | Vibrio and process for producing guluronicacid lyase by using the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920002B (en) * | 2005-08-22 | 2011-06-08 | 中国海洋大学 | Novel vibrionaceae vibrio bacterial strain and application thereof |
| CN106929443A (en) * | 2017-03-13 | 2017-07-07 | 领先生物农业股份有限公司 | A kind of vibrios LX6 2 and its purposes in biological alga fertilizer is prepared |
| CN117362472A (en) * | 2023-11-13 | 2024-01-09 | 山西纳德西生物科技有限公司 | Modified sulfate polysaccharide compound and preparation method thereof |
-
2003
- 2003-11-26 CN CNA2003101057278A patent/CN1544615A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920002B (en) * | 2005-08-22 | 2011-06-08 | 中国海洋大学 | Novel vibrionaceae vibrio bacterial strain and application thereof |
| CN106929443A (en) * | 2017-03-13 | 2017-07-07 | 领先生物农业股份有限公司 | A kind of vibrios LX6 2 and its purposes in biological alga fertilizer is prepared |
| CN117362472A (en) * | 2023-11-13 | 2024-01-09 | 山西纳德西生物科技有限公司 | Modified sulfate polysaccharide compound and preparation method thereof |
| CN117362472B (en) * | 2023-11-13 | 2024-04-19 | 山西纳德西生物科技有限公司 | Modified sulfate polysaccharide compound and preparation method thereof |
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