CN101451113B - Vibrio natriegens and method for producing agarase by using the same - Google Patents
Vibrio natriegens and method for producing agarase by using the same Download PDFInfo
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- CN101451113B CN101451113B CN 200810121997 CN200810121997A CN101451113B CN 101451113 B CN101451113 B CN 101451113B CN 200810121997 CN200810121997 CN 200810121997 CN 200810121997 A CN200810121997 A CN 200810121997A CN 101451113 B CN101451113 B CN 101451113B
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Abstract
The invention relates to novel microbiological Vibrio natriegens CGMCC No.2428 capable of producing agarase and a method for producing the agarase through the strain. The strain is separated from sediments in the East China Sea, belongs to Gram-negative bacteria, is aerobiotic, has an optimum growth NaCl concentration of between 5 and 10 grams per liter and an optimum growth pH value of 7.5, can utilize various sugars such as yeast extract paste, glucose, sucrose and maltose to be energy resources and carbon sources for growth, and belongs to Vibrio natriegens after identification. The V.natriegens CGMCC No.2428 has short growth cycle, and can reach the logarithmic phase by 7 hours; and the strain can induce expression of the agarase, and can collect the agarase from a liquid medium by a simple separation method. The technology for preparing the agarase is simple, vast and quick, has low cost, and can be applied in the field of industrial production or medicine to produce agar oligose.
Description
Technical field
The present invention relates to the new microbe that preserving number is CGMCC No.2428, preservation date is on April 1st, 2008, classification called after vibrios (Vibrio), after be accredited as and need sodium vibrios (Vibrio natriegens), the invention still further relates to the method that this bacterial strain is produced agarase.
Background technology
Agar-agar is a kind of water-soluble polysaccharide in some alginic cell wall, is made up of agarose and agaropectin.Agaropectin mainly is made of substituted radical, baroque polysaccharide chains such as sulfur-bearing acidic group, methyl.Agarose is by (1 → 3)-O-β-D-semi-lactosi and (1 → 4)-O-3, the linear chain molecule that 6-inner ether-α-L-semi-lactosi is alternately formed.
Agarase is a kind of glycosyl hydrolase of the agarose of degrading, and zymogenic bacteria mostly is marine bacteria.Known to inspection information, literature search, some bacterial strain in the genus such as Alterococcus, Alteromonas, Bacillus, Cytophaga, Microscilla, Microbulbifer, Pseudoalteromonas, Pseudomonas, Streptomyces, Zobellia can produce agarase, and Chinese patent 200410023656.1 has been reported a kind of novel bacterial Alteromonas sp.nov.SY37-12 (CCTCCM204009) from the isolating production agarase in marine site, China South Sea.
Character such as that the oligosaccharides that agarase hydrolysis marine alga produces often has is antitumor, antiviral, enhancing immunity can be used as the chiral drug resolution agent and are applied to biomedicine field, promote the industrialization work of biocatalysis and bio-transformation.Algae content is extremely abundant in the ocean; it will be effectively one of source of future-man's based food; the oligose that agarase hydrolysis marine alga produces is used for biomass energy and food; for realizing energy variation; alleviating energy crisis; improve the utility value of existing resource, have great function, and will exert far reaching influence traditional energy and food service industry.In addition, agarase also can be used as the toolenzyme of molecular biological reporter gene and polysaccharide structures mensuration, the zymin of mariculture etc.As seen, agarase can be widely used in fields such as biological medicine, food chemical industry, mariculture as chiral drug resolution agent, biologically active substance production agent, flavour improvers.
Summary of the invention
An object of the present invention is to provide the bacterial strain that a kind of energy production has the enzymatic activity high agarase.
Bacterial strain of the present invention is the new bacterial strain of separating from physical environment, does not also need sodium vibrios (Vibrio natriegens or be abbreviated as V.natriegens) to produce the report of agarase in the past.The need sodium vibrios (V.natriegens) of preparation agarase separates from East Sea settling, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is CGMCC No.2428.
Through identification of morphology and biological characteristic research, preserving number is that the bacterial strain of CGMCC No.2428 has following feature:
(1) colonial morphology: bacterium colony is rounded, smooth surface, and the edge is smooth, projection, oyster white.
(2) cellular form: Gram-negative bacteria, (Olympus BX40) presents mobility, and form is shaft-like (2-3 μ m * 0.4-0.6 μ m) in the majority under the microcytoscope.
(3) physiological and biochemical property: aerobic growth, oxydase, the catalase positive, the indole reaction feminine gender, not hydrolyzed starch, casein, Mierocrystalline cellulose, can utilize yeast extract paste, glucose, pectinose, sucrose, maltose, can not utilize xylan, lactose, sorbyl alcohol, to gentamicin, paraxin sensitivity.
The 16S rRNA gene that to preserving number is the bacterial strain of CGMCC No.2428 carries out pcr amplification and sequencing, find that 16S rRNA Gene Partial fragment length is 532bp, with known to need the 16S rRNA gene order similarity of sodium vibrios (V.natriegens) be 99.4%.The concrete visible sequence table of the 16S rRNA sequence of bacterial strain V.natriegens CGMCC No.2428.
Therefore, content with reference to " Bergey ' s Manual of Systematic Bacteriology " second edition second volume, limiting factor characteristics, morphological specificity and physiological and biochemical index according to bacterial strain, in conjunction with 16S rRNA gene order similarity, identify that bacterial strain CGMCC No.2428 is for needing sodium vibrios (V.natriegens).
Another object of the present invention provides the method that a kind of CGMCC of employing No.2428 produces agarase, may further comprise the steps:
(1) need be seeded in the fermention medium by sodium vibrios (CGMCC No.2428), cultivated 5-10 hour for 20-37 ℃;
(2) in fermention medium, added aseptic agarose inducing culture 15-25 hour;
(3) fermention medium is through high speed centrifugation, and supernatant liquor is through behind the millipore filtration, and rotary evaporation promptly gets agarase to doing.
In the aforesaid method step (1), need the sodium vibrios can be inoculated in the pH value in the fermention medium of 6.5-8.5 (preferred 7.5), under 20-37 ℃ of (preferred 30-35 ℃) temperature, cultivate 5-10 hour (preferred 7 hours), make bacterial strain V.natriegens CGMCC No.2428 reach logarithmic phase.Described fermention medium is composed as follows: sodium-chlor 5-10g/L, and hydrated magnesium chloride 2-6g/L, Repone K 0.5-2.5g/L, Tryptones 5-20g/L, all the other are water, pH is 6.5-8.5 (preferred 7.5).
In the aforesaid method step (2), need to add aseptic agarose inducible strain and express agarase.In substratum, add aseptic agarose to final concentration 0.05-5g/L (being preferably 1g/L), after 150-250 (preferred 180) rpm cultivates 3-6h, rotating speed is adjusted downward to 50-100rpm (preferred 80rpm) and cultivates 15-25h (preferred 18h) again, needing to obtain the enrichment culture liquid of sodium vibrios (V.natriegens).
In the aforesaid method step (3), with the enrichment culture liquid high speed centrifugation in the step (2), for example the centrifugal 5-10min of 5000-10000rpm contains agarase in the supernatant liquor of centrifugal acquisition.Supernatant liquor is by behind the millipore filtration in 0.22 μ m aperture, and rotary evaporation is V.natriegens CGMCC No.2428 agarase to doing, and 4 ℃ of cryopreservations are standby.
The V.natriegens CGMCC No.2428 growth cycle that the present invention finds is short, and 7h just reaches logarithmic phase, this bacterium energy abduction delivering agarase, and the abduction delivering time is 17-22h.By simple separation method, can be from nutrient solution the agarase of collecting high-activity.Agarase preparation technology provided by the invention is simple, with low cost, and is fast a large amount of, is fit to apply to industrial production or field of medicaments, is widely used in fields such as biological medicine, food chemical industry, mariculture to produce agar oligosaccharide.
Description of drawings
Fig. 1 is a V.natriegens CGMCC No.2428 growth curve chart;
Fig. 2 is a V.natriegens CGMCC No.2428 agarase enzyme activity graphic representation under the different agarose inductive conditions;
Fig. 3 is a V.natriegens CGMCC No.2428 agarase enzyme activity graphic representation under the different incubation time conditions
Embodiment
Embodiment 1: the separation screening that needs sodium vibrios (Vibrio natriegens) CGMCC No.2428
Be placed in the old seawater of filtration sterilization that 50ml contains granulated glass sphere picking up from China East Sea sediment sample 2g, vibration is broken up, and gets coating screening culture medium flat board behind the old seawater gradient dilution of supernatant liquid filtering degerming, cultivates observations about 7 days for 37 ℃.Periphery of bacterial colonies forms the bacterial strain of depression and selects preservation, identifies through Lugol's iodine solution, and it is bacterium producing multi enzyme preparation that periphery of bacterial colonies forms the transparent circle person.The inoculation liquid screening substratum that will screening obtains is measured the agarase vigor of fermented liquid, filters out a strain and produces the agarase bacterial strain, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is CGMCCNo.2428.Bacterial strain screening substratum (g/L): sodium-chlor 19.45, magnesium chloride 8.8, calcium chloride 1.8, Repone K 0.55, sodium bicarbonate 0.16, ironic citrate 0.1, Potassium Bromide 0.08, strontium chloride 0.034, boric acid 0.022, sodium hydrogen phosphate 0.008, water glass 0.004, Sodium Fluoride 0.0024, ammonium nitrate 0.0016, yeast extract 5.0, peptone 1.0, pH7.4, all the other are water.
Embodiment 2: the identification of morphology and the biological characteristics that need sodium vibrios (V.natriegens) CGMCC No.2428
V.natriegens CGMCC No.2428 is inoculated in fermention medium (DSMZmedium308), substratum (g/L) composed as follows: sodium-chlor 5.0, and hydrated magnesium chloride 4.0, Repone K 1.0, Tryptones 10.0, pH7.5, all the other are water.The preparation solid medium can add the agar of 20g/L again.Sterilized 30 minutes for 121 ℃.After the inoculation culture, through evaluation, this bacterium has following feature: (1) colonial morphology: bacterium colony is rounded, smooth surface, and the edge is smooth, projection, oyster white.(2) cellular form: Gram-negative bacteria, (Olympus BX40) presents mobility, and form is shaft-like (2-3 μ m * 0.4-0.6 μ m) in the majority under the microcytoscope.(3) physiological and biochemical property: aerobic growth, oxydase, the catalase positive, the indole reaction feminine gender, not hydrolyzed starch, casein, Mierocrystalline cellulose, can utilize yeast extract paste, glucose, pectinose, sucrose, maltose, can not utilize xylan, lactose, sorbyl alcohol, to gentamicin, paraxin sensitivity.
Embodiment 3: the pcr amplification and the sequencing that need the 16SrRNA gene of sodium vibrios (V.natriegens) CGMCC No.2428
V.natriegens CGMCC No.2428 is inoculated in the solid fermentation substratum, and direct picking one ring thalline from the inclined-plane adds 400 μ L sterilized water mixings, 100 ℃ of water-baths 5 minutes, and centrifugal 2 minutes of 12000rpm, supernatant is directly used in PCR.The a pair of universal primer of amplification 16S rRNA gene is as follows:
Forward primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 ';
Reverse primer: 5 '-GGTTACCTTGTTACGACTT-3 ';
Above-mentioned primer is 8-27 and 1510-1492 bit bases of corresponding colibacillary 16S rRNA gene respectively.PCR reaction system (50 μ L) is: 10 * buffer, 5 μ L, 10mMdNTPs1 μ L, each 1 μ L of 4mM primer, ddH
2O 42 μ L, Taq enzyme 0.5 μ L, template DNA 0.5 μ L.The PCR reaction conditions is: 94 ℃ of sex change 45s; 55 ℃ of annealing 45s; 72 ℃ are extended 90s, and 30 circulations are extended 10min for back 72 ℃.PCR product purification and sequencing are finished by Sinogenomax Co., Ltd..The 16S rRNA Gene Partial fragment length of finishing mensuration is 532bp, with bacterial strain V.natriegens ATCC14048
TThe correlated series similarity be 99.4%.The concrete visible sequence table of the 16S rRNA sequence of bacterial strain V.natriegens CGMCC No.2428.
Therefore, content with reference to " Bergey ' s Manual of Systematic Bacteriology " second edition second volume, limiting factor characteristics, morphological specificity and physiological and biochemical index according to bacterial strain, in conjunction with 16S rRNA gene order similarity, identify that bacterial strain CGMCC No.2428 is for needing sodium vibrios (V.natriegens).
Embodiment 4: need sodium vibrios (V.natriegens) CGMCC No.2428 agarase fermentative production condition
V.natriegens CGMCC No.2428 is inoculated in fermention medium (DSMZmedium 308), and its growth scope and optimal growth condition are as follows: the NaCl concentration range is 1-100g/L, and the suitableeest is 5-10g/L; Growth pH value scope is 6.5-8.5, and the suitableeest is 7.5; Growth temperature range is 20-37 ℃, the suitablelyyest is 30-35 ℃.V.natriegensCGMCC No.2428 be inoculated in 35 ℃ of DSMZ medium 308 substratum cultivate 7 hours can be to logarithmic phase (Fig. 1).
Agarose is the inductor of agarase.The aseptic agarose of different concns (0,0.05,0.1,0.2,0.5,1.0,2.0,5.0g/L) added be in the bacterium liquid of logarithmic phase, after 180rpm cultivated 4h, rotating speed was adjusted downward to 80rpm and continues to cultivate 24h.Concentration of reduced sugar is measured according to 3,5-dinitrosalicylic acid (DNS) colorimetry, specifically describe as follows: nutrient solution is through the centrifugal 10min of 5000rpm, directly get 200 μ l supernatant liquors and add isopyknic reaction substrate solution (agarose 2.0g/L, sodium-chlor 5.0g/L, Tris-HCl 50mM, pH7.5), 55 ℃ of reaction 30min behind the abundant mixing of solution add 300 μ l DNS solution boiling water baths colour developing 7min again.Get supernatant 500 μ l behind the centrifugal 5min of solution 2000rpm and be diluted to 2.5ml, quietly to 1min, measure absorbance value (OD
540).Definition enzyme activity unit (U) is: per minute discharges the needed enzyme amount of 1 μ mol reducing sugar.The enzyme activity size of V.natriegensCGMCC No.2428 under different agarose inductive conditions seen Fig. 2, and agarose concentration is under the 5g/L condition, and V.natriegens CGMCC No.2428 agarase output is better than other test conditionss; Agarose concentration is under the 1g/L condition, and V.natriegensCGMCC No.2428 agarase output can reach 10.3U/ml.
Aseptic agarose added be in the bacterium liquid of logarithmic phase, to agarose concentration be 1g/L and 5g/L, after 180rpm cultivated 4h, rotating speed is adjusted downward to 80rpm to be continued to cultivate.Every the 4-8h sampling, measure agarase enzyme size alive (Fig. 3) in the supernatant liquor.Agarose abduction delivering 17-22h, V.natriegens CGMCC No.2428 agarase output can reach maximum value, is 17-20U/ml.
Embodiment 5: need the preparation of sodium vibrios (Vibrio natriegens) CGMCC No.2428 agarase
Add aseptic agarose in the V.natriegens CGMCC No.2428 bacterium liquid of logarithmic phase to final concentration 1g/L, after 180rpm cultivated 4h, rotating speed was adjusted downward to 80rpm and cultivates 18h.The centrifugal 10min of solution 5000rpm, supernatant liquor are by behind the millipore filtration in 0.22 μ m aperture, and 40-45 ℃ of rotary evaporation is V.natriegens CGMCCNo.2428 agarase to doing, and 4 ℃ of cryopreservations are standby.In the use, adopt Tris-HCl-NaCl solution (sodium-chlor 5.0g/L, Tris-HCl50mM, pH=7.5) dissolving, add to the agarose solution mixing after 55 ℃ of temperature bathe 12-24h, can produce agar oligosaccharide.
Sequence table
＜110〉The Second Institute of Oceanograghy,SOA
＜120〉a kind of method that needs the sodium vibrios and adopt this bacterium production agarase
<160>1
<170>PatentIn?version3.3
<210>Seq?ID?No.1
<211>532
<212>DNA
<213>Vibrio?natriegens
<400>Seq?ID?No.1
Claims (10)
1. a need sodium vibrios (Vibrio natriegens), culture presevation number is CGMCCNo.2428.
2. need sodium vibrios (Vibrio natriegens) according to claim 1 is characterized in that: the described 16S rRNA sequence of sodium vibrios (Vibrio natriegens) that needs is shown in Seq ID NO.1.
3. one kind is utilized the described method that needs sodium vibrios (Vibrio natriegens) to prepare agarase of claim 1, comprises the steps:
(1) need be seeded in the fermention medium by sodium vibrios CGMCC No.2428, cultivated 5-10 hour for 20-37 ℃;
(2) in fermention medium, added aseptic agarose inducing culture 15-25 hour;
(3) fermention medium is through high speed centrifugation, and supernatant liquor is through behind the millipore filtration, and rotary evaporation promptly gets agarase to doing.
4. method according to claim 3 is characterized in that, the fermention medium in the described step (1) comprises following composition: sodium-chlor 5-10g/L, hydrated magnesium chloride 2-6g/L, Repone K 0.5-2.5g/L, Tryptones 5-20g/L, all the other are water, and pH is 6.5-8.5.
5. method according to claim 4 is characterized in that: the fermention medium in the described step (1) comprises following composition: sodium-chlor 5g/L, and hydrated magnesium chloride 4g/L, Repone K 1g/L, Tryptones 10g/L, all the other are water, pH is 7.5.
6. method according to claim 3 is characterized in that, the culture temperature in the described step (1) is 30-35 ℃.
7. method according to claim 3 is characterized in that, adds aseptic agarose to final concentration 0.05-5g/L in the described step (2) in fermention medium.
8. method according to claim 7, it is characterized in that, the process of described step (2) is: add aseptic agarose in the substratum to final concentration 0.05-5g/L, after 150-250rpm cultivates 3-6h, rotating speed is adjusted downward to 50-100rpm, cultivate 15-18h again, needing to obtain the enrichment culture liquid of sodium vibrios (V.natriegens).
9. method according to claim 3 is characterized in that, the centrifugal speed in the described step (3) is 5000-10000rpm.
10. method according to claim 3 is characterized in that, the millipore filtration aperture in the described step (3) is 0.22 μ m.
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| CN104611312A (en) * | 2015-02-16 | 2015-05-13 | 集美大学 | Culture medium for producing agarase by virtue of fermentation of vibrio natriegen and fermentation method for producing agarase by virtue of fermentation of vibrio natriegen |
| CN105087427A (en) * | 2015-07-06 | 2015-11-25 | 华侨大学 | Vibrio natriegens for producing agarase and application of vibrio natriegens |
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| CN101955895B (en) * | 2010-02-09 | 2012-05-23 | 浙江工业大学 | Roseobactersp.zjut and application thereof in preparation of agaro-oligosaccharide |
| CN101845447B (en) * | 2010-04-22 | 2012-04-04 | 国家海洋局第二海洋研究所 | Beta-agarase encoding gene and gene acquisition method |
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| CN105087427B (en) * | 2015-07-06 | 2018-08-24 | 华侨大学 | Produce Vibrio natriegen and its application of agarase |
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