CN1920002B - Novel vibrionaceae vibrio bacterial strain and application thereof - Google Patents
Novel vibrionaceae vibrio bacterial strain and application thereof Download PDFInfo
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- CN1920002B CN1920002B CN200510090973XA CN200510090973A CN1920002B CN 1920002 B CN1920002 B CN 1920002B CN 200510090973X A CN200510090973X A CN 200510090973XA CN 200510090973 A CN200510090973 A CN 200510090973A CN 1920002 B CN1920002 B CN 1920002B
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- bacterial strain
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention relates the vibrionaceae vibrio strain and its application. The invention relates the vibrionaceae vibrio strain from gulfweed, and the output of algin lyase made by the vibrionaceae vibrio strain is 2-37 times than that of algin lyase made with now technology. The algin lyase can be used to make algin oligosaccharide.
Description
Technical field
The present invention relates to microorganism field.Particularly, the present invention relates to a kind of new vibrionaceae vibrio bacterial strain Vibrio sp.QY2, its deposit number is CCTCC NO:M205031, and the application for preparing algin catenase and algin oligosaccharide with this bacterial strain.
Background technology
Algin is that a kind of source is abundant, broad-spectrum acid marine polysaccharide, derive from brown alga and some bacterium (comprising pseudomonas, vinelandii etc.), pass through β-1 by beta-D-mannuronic acid and α-L-guluronic acid, 4 glycosidic links are connected to form, and can be divided into polymannuronic acid, poly-guluronic acid and mannuronic acid and guluronic acid and replace block.Recently, the biological activity of algin oligosaccharide constantly is found, and as antitumor, antiviral, antibiotic, anti-cardiovascular disease etc., its wide application prospect has caused people's extensive concern.Algin catenase can interrupt the β-1 of algin by β cancellation mechanism, 4 glycosidic links, and at the non-reduced terminal C4 that produces, 5 unsaturated double-bonds, the generation of these reduction end unsaturated double-bonds has material impact to some biological activity of oligosaccharides, and the product of physics, chemical degradation method degraded does not have these characteristics.Enzyme process prepare algin oligosaccharide single because of the gentle easily control of reaction conditions, product, do not cause characteristics such as environmental pollution to replace acid system gradually.Now be separated to tens of kinds of algin catenases from bacterium, invertebrates, yield of enzyme is low, the shortcoming of substrate specificity difference but ubiquity, and has limited the application of algin oligosaccharide greatly.And the present inventor produces the marine bacteria CCTCC M 205031 of algin catenase outside the born of the same parents from the sargassun surface isolation to a plant height, and yield of enzyme is than exceed 2～37 times of having reported, and separation obtains a kind of algin catenase from its tunning.
Summary of the invention
One of purpose of the present invention provides a kind of new vibrionaceae vibrio bacterial strain (Vibrio sp.QY2) CCTCC NO:M205031.
Another object of the present invention provides a kind of algin catenase that utilizes the above-mentioned bacterial strains preparation.
A further object of the present invention provides the algin oligosaccharide that utilizes above-mentioned algin catenase preparation.
According to a technical scheme of the present invention, from the sargassun surface isolation obtain a kind of new vibrionaceae vibrio bacterial strain (Vibrio sp.QY2, it is preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M205031.
According to another technical scheme of the present invention, the invention provides a kind ofly by fermentation above-mentioned bacterial strains, the algin catenase that obtains of separation and purification then, its molecular weight is 28.5kD.
According to a technical scheme more of the present invention, the invention provides a kind of by using above-mentioned algin catenase to decompose the algin oligosaccharide that algin obtains.
Vibrios CCTCC M 205031 of the present invention can be used in the biology preparation of algin catenase, and its yield of enzyme exceeds 2～37 times than the yield of enzyme of the bacterial strain of having reported.
Vibrionaceae vibrio bacterial strain of the present invention (Vibrio sp.QY2) CCTCC M 205031, be preserved in (address: Chinese Wuhan Wuhan University, Chinese typical culture collection center on April 7th, 2005, postcode: 430072), deposit number is CCTCC NO:M205031.
Description of drawings
Fig. 1 is the mass spectrum according to the brown alga glycocoll of embodiments of the invention 4 preparations.
Embodiment
The separation of embodiment 1 vibrios CCTCC M 205031
Get sargassun 1g, be dipped in 100 milliliters of sterilization seawater, stirred 1 hour, get 1 milliliter of suspension and insert 100 milliliters of liquid substratum, 25 ℃ of static one weeks of cultivation.Get nutrient solution and on solid plate, rule and separate mono-clonal, continuous 5 generations the mono-clonal purifying cultivate.The picking mono-clonal is inoculated in the test tube that contains 3 milliliters of liquid substratum, and 25 ℃ of 200r/min cultivated 12 hours.Nutrient solution inserted in 1% ratio contain in 250 milliliters of triangular flasks of 50 milliliters of liquid substratum, cultivated 3 days in 25 ℃ of 200r/min.Nutrient solution is removed thalline in 4 ℃ of centrifugal 10min of 12000r/min, measures the enzyme activity of supernatant liquor, chooses the highest bacterial strain of enzyme activity.Wherein the enzyme activity of strain fermentation using bacteria generation is the highest, is 1208U/L, with its called after QY2.The liquid culture based formulas of using is (g/L): sodium alginate 3.0g, KH
2PO
43.0g, K
2HPO
47.0g, (NH
4)
2SO
42.0g, MgSO
40.1g, FeSO
40.1g, NaCl30.0g; Add the agar of 12.0g/L in the solid medium.Enzyme activity determination adopts ultraviolet absorption method, gets 0.9 milliliter of 3g/L sodium alginate (with the phosphate buffered saline buffer preparation of 0.1mol/L pH7.0), adds 0.1ml enzyme liquid, 40 ℃ of insulation 15min, and the assaying reaction system is at the absorbance value at 235nm place.With this understanding, per minute absorbance value increase by 0.1 is an enzyme activity unit (U).
The evaluation of embodiment 2 vibrios CCTCC M205031
1, morphological specificity
Bacterial strain CCTCC M 205031 cultivates the bacterium colony of 24h on conventional Zobell 2216E flat board rounded, translucent, smooth surface, and neat in edge, diameter is 1.5mm; Bacterium colony is yellow on conventional TCBS substratum, and diameter is 2.1mm.This bacterial strain is Gram-negative, and is shaft-like, movable.
2, growth conditions
Bacterial strain CCTCC M 205031 is a facultative anaerobe.Bacterial strain CCTCC M 205031 well-grown in 1%～7%NaCl concentration range, the suitableeest NaCl concentration is 3%, 25 ℃ of optimum growth temperatures, the suitableeest growth pH6.0.
3, physiological and biochemical property
Routine biochemistry experiment shows, the bacterial strain CCTCC M 205031 oxydase reaction positives, glucose fermentation produces not aerogenesis of acid, to vibrios inhibitor O/129 (2,4-diamino-6, the 7-di-isopropyl pyridine phosphoric acid salt of talking endlessly, 150mg/L) sensitivity, not luminous, do not produce pigment.The combining form feature judges that tentatively bacterial strain CCTCC M 205031 belongs to vibrionaceae vibrio bacterial (Vibrio sp.).
4, the biology classification position determines
Morphological specificity with bacterial strain CCTCC M 205031, the feature of physiological and biochemical property and Vibrio part bacterium compares, result's (table 1) shows, bacterial strain CCTCC M 205031 is the most approaching with Vibrio aestuarianus (Vibrio aestuarianus), only at arginine dihydrolase, V-P produces, 40 ℃ of 3 aspect differences such as growth, with Vibrio splindidus (Vibrio splendidus) at arginine dihydrolase, reduction nitrate, luminous, the pectinose fermentation, amylase produces, gelatinase produces, 7 aspect differences such as chitinase generation, with dinitrogen vibrios (Vibriodiazotrophicus) at arginine dihydrolase, reduction nitrate, the seminose fermentation, lactose fermentation, the saligenin fermentation, amylase produces, 7 aspect differences such as lipase generation are being moved about with vibrio alginolyticus (Vibrio alginolyticus), reduction nitrate, V-P produces, 4 ℃ of growths, the pectinose fermentation, the inositol fermentation, amylase produces, gelatinase produces, molten algae enzyme produces, 10 aspect differences such as chitinase generation.
The comparison of table 1 bacterial strain CCTCC M 205031 and Vibrio part member phenotypic characteristic
+, positive more than 90% bacterial strain;-, negative more than 90% bacterial strain; D, 11～89% bacterial strain is positive.
Bacterial strain CCTCC M 205031 is gram negative bacillus, and is movable, the oxydase reaction positive, glucose fermentation produces not aerogenesis of acid, and is to vibrios inhibitor O/129 sensitivity, not luminous, do not produce pigment, therefore preliminary judgement bacterial strain CCTCC M 205031 belongs to vibrionaceae vibrio bacterial (Vibriosp.).Comparison according to the physiological and biochemical property of this bacterial strain and part Vibrio bacterium, CCTCC M205031 and V.aestuarianus are the most approaching, but it is to be noted, in uncle's Jie Shi Bacteria Identification handbook (the 9th edition), have many physiological and biochemical properties not describe, so CCTCC M 205031 might also have more similarities and differences with V.aestuarianus for V.aestuarianus.The preparation of embodiment 3 algin catenases
(1) yeast culture
The mono-clonal of picking vibrios CCTCC NO:M205031 is inoculated in the test tube that contains 3 milliliters of liquid substratum, and 25 ℃ of 200r/min cultivated 12 hours.Nutrient solution inserted in 1% ratio contain in 250 milliliters of triangular flasks of 50 milliliters of liquid substratum, cultivated 3 days, collect nutrient solution in 25 ℃ of 200r/min.The liquid culture based formulas of using is (g/L): sodium alginate 3.0g, KH
2PO
43.0g, K
2HPO
47.0g, (NH
4)
2SO
42.0g, MgSO
40.1g, FeSO
40.1g, NaCl30.0g.
(2) separation and purification of enzyme
Nutrient solution is removed thalline in 4 ℃ of centrifugal 10min of 12000r/min, collects supernatant liquor.Slowly add ammonium sulfate to 80% saturation ratio in supernatant liquor, 4 ℃ leave standstill 3h, 4 ℃ of centrifugal 30min of 13000g, abandoning supernatant, precipitation is dissolved in 40mmol/L pH7.0 phosphate buffered saline buffer, 4 ℃ of dialysed overnight, 4 ℃ of centrifugal 30min of 13000g get supernatant liquor, are crude enzyme liquid.Crude enzyme liquid is splined on 40mmol/L pH7.0 phosphate buffered saline buffer equilibrated DEAE Sepharose FastFlow post and carries out ion exchange chromatography.The chromatography column volume is that (1cm * 20cm), applied sample amount is 3ml to 14ml, and with the damping fluid that contains 0.4-0.6mol/L NaCl gradient elution stage by stage, flow velocity 1.0ml/min detects the gleanings activity, collects active ingredient in a large number.Active ingredient is carried out gel permeation chromatography, Superdex 75 HR10/30 posts 20mmol/L pH7.0 phosphate buffered saline buffer balance, applied sample amount 1.0ml, with the balance liquid wash-out, flow velocity 0.5ml/min, detection gleanings activity, a large amount of collecting high-activity components concentrate standby (being called " enzyme liquid " later on).Above chromatography purification operation is all carried out at 4 ℃.Pressing Folin-phenol method, is proteinic concentration in the standard test enzyme liquid with the bovine serum albumin, and its concentration is 1.77mg/mL, is 276U/mg than vigor.
Vibrios CCTCC NO:M205031 yield of enzyme is 1208U/L, is 2～37 times of the strain enzyme-producing amount reported.
The SDSPAGE analytical results shows that the molecular weight of this enzyme is about 28.5kD.
Enzyme liquid is incubated 1h down at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ respectively, be cooled to-20 ℃ rapidly, measure enzymic activity at 40 ℃ by ultraviolet absorption method then, simultaneously, measure the activity of enzyme liquid under differing temps respectively, to determine the optimum temperuture of enzyme reaction.The result shows that the optimum temperuture of enzyme reaction is 40 ℃; This algin catenase is stable when being lower than 40 ℃, and when temperature was higher than 40 ℃, the vigor of enzyme was decayed rapidly.
Measure enzymic activity under different pH, enzymic activity is the highest when finding about pH7.1; This enzyme (pH5～10) stability in the larger context is higher, and enzymic activity all remains on more than 80%.
With the enzyme work that does not add any metal ion is 100%, Mg
2+, Ca
2+Activity for enzyme has promoter action, and Zn
2+, Ba
2+, Ni
2+, Al
3+And EDTA, SDS have restraining effect for the activity of enzyme.
Use poly (M) and poly (G) that the substrate specificity of enzyme is carried out preliminary study, the result shows that this algin catenase is higher than activity to poly (G) to the activity of poly (M), and its relative reactivity ratio is 100: 47.
The preparation of embodiment 4 algin oligosaccharides
Sodium alginate is made into the aqueous solution that concentration expressed in percentage by weight is 1-5%, every liter of solution adds the above-mentioned algin catenase of 10-50U/L, at 25-40 ℃ of incubation 8-16 hour, at last by centrifugal or remove by filter undegradable macromole algin, carry out chromatographic separation with Sephadex G25 gel column, collect the algin oligosaccharide component, concentrate drying.Can obtain the algin oligosaccharide that the polymerization degree is 3-6.
Mass spectrum is identified the composition of brown alga oligose, and used mass spectrum is MALDI ToF massspectrometer (BIFLEX IIIBruker Daltonics), is finished by Chinese Academy of Sciences chemistry institute mass spectrum center.Analyze MALDI ToF mass spectroscopy collection of illustrative plates as can be known: 551.38 peak ownership is trisaccharide, and 727.51 peak ownership is tetrose, and 903.64 peak ownership is pentasaccharides, and 1079.76 peak ownership is six sugar.Can calculate each components contents according to peak intensity, trisaccharide content is 77.8%, and tetrose content is 16.5%, and pentasaccharides content is that 3.6%, six sugared content is 2.1%.
Claims (3)
1. a vibrionaceae vibrio bacterial strain (Vibrio sp.) QY2, its preserving number is CCTCCNO:M 205031, using the algin catenase of this bacterial strain preparation to decompose algin, to obtain the polymerization degree be 3～6 algin oligosaccharide.
2. the algin catenase of a bacterial strain QY2 preparation of using claim 1.
3. use the described algin catenase of claim 2 to prepare the purposes that the polymerization degree is 3～6 algin oligosaccharide.
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| CN1920002B true CN1920002B (en) | 2011-06-08 |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591628B (en) * | 2009-06-11 | 2011-05-25 | 浙江工业大学 | Acinetobacter juni. X8 and application thereof in preparing algin lyase |
| CN102311930B (en) * | 2010-07-02 | 2015-10-07 | 中国海洋大学 | A kind of algin catenase produced by Pseudomonas.sp.HZJ216 |
| CN101993848B (en) * | 2010-11-08 | 2012-11-21 | 淮海工学院 | Ocean low-temperature dextranase, enzyme producing method and strain S6-2 produced therefrom |
| CN102994407B (en) * | 2011-12-16 | 2014-10-29 | 中国科学院大连化学物理研究所 | Flavobacterium strain and incision alginate lyase coding gene, preparation and application |
| CN102586216B (en) * | 2012-03-15 | 2014-10-08 | 华东理工大学 | Method for producing sodium alginate lyase by utilizing vibrio vulnificus |
| CN103565817A (en) * | 2013-11-11 | 2014-02-12 | 深圳大学 | Application of alginate oligosaccharide |
| CN105624137B (en) * | 2014-11-18 | 2020-01-21 | 中国科学院大连化学物理研究所 | Alginate lyase Algb and coding gene and application thereof |
| CN106701627B (en) * | 2017-01-05 | 2020-07-03 | 中国海洋大学 | Marine vibrio with high yield of alginate lyase and application thereof |
| CN110272852B (en) * | 2019-07-24 | 2021-09-28 | 江南大学 | Vibrio natriegens and application thereof |
| CN114657085B (en) * | 2021-12-24 | 2023-10-20 | 中国热带农业科学院热带生物技术研究所 | High-yield algin lyase strain and application thereof |
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|---|---|---|---|---|
| CN1380342A (en) * | 2001-04-07 | 2002-11-20 | 青岛海洋大学 | Production method of phycocolloid oligosaccharide |
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