CN108130283A - A kind of bacillus of degradable Microcystin and its application - Google Patents
A kind of bacillus of degradable Microcystin and its application Download PDFInfo
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- CN108130283A CN108130283A CN201711106728.2A CN201711106728A CN108130283A CN 108130283 A CN108130283 A CN 108130283A CN 201711106728 A CN201711106728 A CN 201711106728A CN 108130283 A CN108130283 A CN 108130283A
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Abstract
The present invention relates to a kind of bacillus of degradable Microcystin, the bacillus is preserved in China typical culture collection center on June 26th, 2017, and preserving number is CCTCC NO:M2017381；Further relate to application of the bacillus in degrading microcystic toxins and the application in the composition for preparing degradable Microcystin；Further relate to the composition of degradable Microcystin prepared by a kind of method of composition for preparing degradable Microcystin and this method.By bacillus of the invention and the composition prepared using the bacillus, the Microcystin in the water body that can effectively degrade purifies water, prevents water environment degradation, protects water ecology.
Description
Technical field
The present invention relates to the microorganism of degrading microcystic toxins and method field, more specifically it relates to a kind of degradable micro-
The bacillus of capsule algae toxin and its application.
Background technology
With the rapid development of human being's production, life style, industrialize, the progress faster of urbanization, it is a large amount of containing abundant
The industrial wastewater and sanitary sewage of nitrogen and phosphorus pollution object are discharged into water body, and water pollution and eutrophication problem are on the rise.At me
Frequently there is cyanobacterial bloom outburst in state's freshwater lake such as Dian Chi, Taihu Lake, Chaohu and East Lake etc..
Wawter bloom can all cause tremendous influence to the mankind and natural environment.First, wawter bloom algae is kept afloat, and makes water body
Transparency is substantially reduced, and smell becomes stench unpleasant, and the photosynthesis of algae is significantly suppressed, reduces dissolved oxygen source, algae
It rots to decompose after class death, also consumes a large amount of dissolved oxygens of water body, lead to the aquatic animals and plants such as fish in water body due to lacking oxygen
And it is dead, the ecosystem will be caused disorderly when serious.Secondly, according to research reports, after the especially Microcystis aeruginosa death of some algae
Algae toxin can be released.In wawter bloom, the burst size of algae toxin is huge, and harm is serious, not only jeopardizes water ecology, also jeopardizes people
Class reference water security.
Toxicologic study finds that, by the extract of algae and pure Microcystin, oral, intraperitoneal injection can cause liver
Popular name for becomes.No matter what difference the germline of animal, gender, age have, acute, subacute and chronic toxicity test and it is in vitro, in body
In experiment, Microcystin shows toxicity similar, high special, using liver as target organ.Microcystin into
After entering liver cell, the activity of energy strong inhibition phosphoprotein phosphatase, and inhibit protein phosphatase 1 type (PP1) and albumen in specific manner
The activity of phosphatase 2A types (PP2A) correspondingly increases the activity of protein kinase, leads to the mistake of intracellular multiple proteins
Phosphorylation breaks intracellular protein phosphorylation and the balance of dephosphorylation.This life is further amplified by cell signaling system
Change effect, change the activity of a variety of enzymes, cause intracellular a series of biochemical reactions disorderly, cause micro- in cytoplasm
Wire network is reset, and eucaryotic cell structure is destroyed with overall stability, and liver cell is caused to deform.Microcystin toxicological effect it is main
Target organ is liver, with the progress in relation to Microcystin toxicity, also there is multiple organ toxicity, genetoxic and carcinogenic
The relevant report of property.Other than inhibiting phosphatase intoxicating, oxidative stress and Mitochondria permeability transition are caused in Microcystin
Also critically important effect is played in malicious mechanism.Microcystin can be generated with active oxygen in inducing cell (ROS), lead to cell
Damage and lipid peroxidation, and possibly through certain access inducing cell apoptosis, the hepatotoxicity part of Microcystin
It is caused by the generation of induced activity oxygen (ROS).
Microcystin property is stablized, and fusing point is high, can still keep active structure at 300 DEG C.The easy stabilizing dissolved of algae toxin
In water or methanol, remove by physical methods such as coagulation, precipitation, filterings the Microcystin being dissolved in water.
Algae toxin chemical property is also more stable, insensitive to soda acid variation, need to use the strong oxidizers such as ozone, chlorine, potassium permanganate
Just it is remarkably improved the degradation rate of algae toxin.The rock-steady structure of algae toxin can still be kept under ultraviolet light, but ultraviolet light is for a long time
In the case of irradiation, algae toxin content in water can be reduced.
Research shows that it can effectively remove algae toxin using the method for physics and chemistry.It is more common in physical method to have
The methods of activated carbon adsorption, coagulating sedimentation, ultrafiltration, nanofiltration, reverse osmosis membrane.These physical methods can with rapid, high volume remove
Frustule and algae toxin, but its is of high cost, is not easily recycled, and algae toxin removes the shortcomings of incomplete, restricts the hair of Physical
Exhibition.For chemical method using the strong oxidizers such as ozone, chlorine, potassium permanganate removal algae toxin, remaining chemical substance can be right
Water body generates the influence of secondary pollution.While can cause to rely on the damage of the animals and plants of this piece waters life, finally destroy water body
The ecological balance.
Studies have found that some bacteriums can generate the substance for decomposing Microcystin.However, this being separately cultured at present
Class bacterium is seldom, and majority rests on laboratory stage.Therefore, it is necessary to one kind can in natural water efficient degradation micro-capsule
The bacterium of algae toxin or other microorganisms.
Invention content
Inventor detaches, obtaining one plant can be with Microcystis aeruginosa from Zhengzhou Wastewater Sample through Microcystin screening and culturing
The bacterial strain that toxin is sole carbon source and nitrogen source is grown.The bacterial strain is dyed in Gram-positive, and acid-fast stain is negative, energy
Generate gemma.It extracts its genome and carries out 16 S rDNA sequencings, sequence such as SEQ ID NO:Shown in 1, by the sequence in ncbi
It is compared in database, discovery is higher with the sequence degree of approximation of bacillus (Bacillus sp.), is named as
Bacillus subtilis MCS1 (Bacillus subtilis MCS1).
It is found based on above, the present invention provides a kind of bacillus of degradable Microcystin, the bacillus
China typical culture collection center is preserved on June 26th, 2017, preserving number is CCTCC NO:M2017381.
The present invention also provides application of the above-mentioned bacillus in degrading microcystic toxins.
The present invention also provides application of the above-mentioned bacillus in the composition for preparing degradable Microcystin.
The present invention also provides a kind of method for the composition for preparing degradable Microcystin, including from above-mentioned bud
The step of spore bacillus extracts to obtain the composition of the degradable Microcystin.
In a specific embodiment, it the described method comprises the following steps：
S1：It is inoculated with and cultivates the bacillus and obtain bacillus culture, the bacillus culture is concentrated
Into concentration liquid；
S2：The concentration liquid is crushed, obtains bacterial cell disruption liquid；
S3：The bacterial cell disruption liquid is centrifuged, takes supernatant to get to the composition of the degradable Microcystin.
Southern in a specific embodiment, the state in S1 before the bacillus is inoculated with is less than -20 DEG C of temperature
When freezing state under degree, before inoculated and cultured, the bacillus is activated.
In a preferred embodiment, by centrifuging so bacillus culture collects thalline, and uses corpusculum in S1
The thalline is resuspended to carry out the concentration in the phosphate buffer of long-pending 50mmol/L.
In a preferred embodiment, in S2, the method by ultrasonication will crush.
In a preferred embodiment, step S4 is further included after S3：Combination to the degradable Microcystin
Protein concentration in object is quantified.
The present invention also provides a kind of compositions of degradable Microcystin being prepared by above method matter.
By bacillus of the invention and the composition prepared using the bacillus, in the water body that can effectively degrade
Microcystin, purify water, prevent water environment degradation, protect water ecology.
Microbial preservation
The bacterial strain of the present invention being related to is isolated from Zhengzhou Wastewater Sample, through 16S rDNA sequencing, morphology and
Other Characters Identifications, the bacterial strain belong to bacillus, and 16S rDNA sequences and the Bacillus niabensis degrees of approximation
Compare high.The bacterial strain is preserved in the Chinese Typical Representative culture of Wuhan University of Wuhan City of Hubei China province on June 26th, 2017
Collection (CCTCC), preserving number are CCTCC NO:M2017381 is named as bacillus subtilis MCS1 (Bacillus
subtilis MCS1)。
Although it should be noted that inventor in preservation by the strain was named bacillus subtilis MCS1
(Bacillus subtilis MCS1), however the name be used only for descriptive effect rather than by the bacterial strain fixed kind be withered
Careless bacillus (Bacillus subtilis), and the 16S rDNA sequences of the bacterial strain and the consistency of bacillus subtilis
It is not high, it is not clustered (Fig. 1) together both in cluster analysis.Combining form feature and 16S rDNA sequences, the bacterial strain
There are difference with the Bacillus that is currently known, and therefore, which in fact belongs to new kind.
Description of the drawings
Fig. 1 is the situation for adding in the culture mediums of different water samples after culture 3 days, and A is control group, and B is East Lake water sample group, C
For Zhengzhou water sample group；
Fig. 2 be the present invention bacillus 16S rDNA sequences and 15 in existing bacillus 16S rDNA sequences into
The systematic evolution tree that row cluster obtains；
Fig. 3 is influence of the different temperatures to degradation of microcystins efficiency；
Fig. 4 is influences of the different pH to degradation of microcystins efficiency；
Fig. 5 is influence of the different protein concentrations to degradation of microcystins efficiency.
Specific embodiment
The principle of the present invention and feature are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. the screening separation of the bacillus of degradable Microcystin
The extraction of Microcystin：The Microcystin used in following embodiment extracts from microcystic aeruginosa (FACHB-
905, aquatile research institute of the Chinese Academy of Sciences).
Sampling：Inventor acquires water sample from Zhengzhou sewage plant and Wuhan East Lake, using aseptic double-distilled water as control, respectively
It is inoculated in beef-protein medium, continuous 3 times, each 3d is carried out under the conditions of natural lighting, 37 DEG C, 220r/min
Shaking flask enrichment culture.Then this enrichment culture liquid dilution certain multiple is being inoculated in using algae toxin as sole carbon source and nitrogen
In the M9 culture mediums in source, growing state is observed in shaking flask culture 3-4 days.The results show that it is vaccinated with the water from Zhengzhou pond
Sample becomes apparent muddy (Fig. 1) by sole carbon source and the M9 culture mediums of nitrogen source of algae toxin after shaking flask culture, thus it is speculated that wherein
There are can be using algae toxin as sole carbon source and the algal toxin degradation bacterium of the growth of nitrogen source progress.
After the tablet culture of three days, the tablet for being coated with Wuhan water sample and control group does not have bacterium colony to grow, and applies
Being furnished on the tablet of Zhengzhou Wastewater Sample has bacterium colony to grow.Tablet cultivation results further illustrate, are not present in the water sample of Wuhan
Algal toxin degradation bacterium is tentatively judged in the water sample of Zhengzhou there are algal toxin degradation bacterium, and according to the form of bacterium colony, in the water sample of Zhengzhou
There are at least two kinds of algal toxin degradation bacterium.
One of which bacterium is similar to bacillus from morphology and other characters, and Gram's staining result is in sun
Property, and gemma can be generated.The bacterial strain was preserved on 2 22nd, 2017 in Wuhan University of Wuhan City of Hubei China province
State's Type Tissue Collection (CCTCC), preserving number are CCTCC NO:M2017381 is named as bacillus subtilis MCS1
(Bacillus subtilis MCS1).The character of the bacterial strain is as shown in table 1,16S rDNA sequences such as SEQ ID NO:1 institute
Show.The 16S rDNA sequences and the homology of bacillus are higher, by the sequence further with 15 bacterium of bacillus
Strain carries out sequence alignment, and phylogenetic tree construction (Fig. 2), the results show that the bacterial strain can be included into bacillus
(Bacillus.sp)。
The character of 1 bacillus subtilis MCS1 of table (Bacillus subtilis MCS1)
2. the extraction of the composition of degradable Microcystin
1) inoculated and cultured：- 20 DEG C of separated bacterial strains above frozen are taken, are inoculated in the examination equipped with 3ml LB culture mediums
Guan Zhong, the shaken cultivation at 37 DEG C, until culture is transferred in the 500ml tapers equipped with 300 ml LB culture mediums after logarithmic phase
In bottle, the shaken cultivation at 37 DEG C.
2) centrifugal concentrating：After culture is to stationary phase, refrigerated centrifuge is carried out, 9000r/min centrifuges 15min at 4 DEG C.
The clear 5ml of phosphate buffer of 50mmol/L is taken, cell suspension is made in cleaning thalline 3 times.
3) ultrasonication, broken condition are carried out to above-mentioned cell suspension：Output power 300W, ultrasonic 30min (open by ultrasound
8s closes 4s), ice bath.The primary ultrasonic step is carried out after the 30min of interval again.
4) bacteria breaking liquid carries out refrigerated centrifuge separation.4 DEG C, rotating speed 8000r/min of temperature centrifuges 20min, institute's supernatant
For intracellular organic matter --- acellular intracellular crude enzyme liquid (that is, composition of degradable Microcystin) is placed in -20 DEG C of refrigerators
In save backup.
Protein content in the composition of degradable Microcystin is measured using BCA determination of protein concentration kit.
3. the optimization of degradation of microcystins condition
Influence of 3.1 temperature to degradation efficiency
With the 0.05mol/L phosphate buffered saline reaction systems of pH 7.4, Microcystin and degradable micro- is added
The composition of capsule algae toxin, the initial concentration for making Microcystin is 10mg/l, and the composition of degradable Microcystin causes
Protein concentration be 280mg/l.Oscillation incubation, rotating speed 150r/min at 25,30,35 DEG C respectively.It is being incubated certain time
Afterwards, the Microcystins Concentration in reaction system is detected, and is converted into degradation efficiency to embody temperature to the degradable of the present invention
The influence of the efficiency of the composition degradation algae toxin of Microcystin.
The results are shown in Figure 3, and with the raising of temperature, the degradation efficiency of Microcystin is gradually increased.35 DEG C of conditions
Under, enzymatic reaction is fastest.5 hours degradation efficiencies reached 70% or so up to 60% or so, 15 hour degradation efficiency, but
Reaction rate under the conditions of subsequent 35 DEG C is begun to decline, and is 80% or so to 25 hours degradation efficiencies, under the conditions of 25 and 30 DEG C
Reaction tendency is similar, and degradation efficiency is slow at the beginning, and the degradation efficiency of 5 hours is but anti-later in 30-40% or so
Rate is answered gradually to accelerate also to reach 80% or so to degradation efficiency at 25 hours.As it can be seen that if short time treatment, temperature is best
35 DEG C are adjusted to, if being handled when long, temperature is adjustable as 25-35 DEG C.
Influences of the 3.2pH to degradation efficiency
With 0.05mol/L phosphate buffered saline reaction systems, it is respectively 6.0,7.0 and 8.0 to adjust pH.It adds micro-
The composition of capsule algae toxin and degradable Microcystin makes the initial concentration of Microcystin for 10mg/l, degradable micro-capsule
Protein concentration caused by the composition of algae toxin is 280mg/l..The oscillation incubation at 30 DEG C, rotating speed 150r/min.It is being incubated
After a certain period of time, the Microcystins Concentration in reaction system is detected, and is converted into degradation efficiency to embody pH to the present invention's
The influence of the efficiency of the composition degradation algae toxin of degradable Microcystin.
The results are shown in Figure 4, and when pH is 8,5 hours degradation efficiencies are more than 60%, and final degradation efficiency is 80% or so.
When pH is 7,5 hours degradation efficiencies are 50% or so, and final degradation efficiency is 80% or so.Therefore, can be 7-8 by pH.
Influence of 3.3 composition concentrations to degradation efficiency
With 0.05mol/L phosphate buffered saline reaction systems, Microcystin and degradable Microcystin are added
Composition, making the initial concentration of Microcystin, albumen caused by the composition of degradable Microcystin is dense for 10mg/l
It spends for 140mg/l, 280mg/l and 350mg/l.The oscillation incubation at 30 DEG C, rotating speed 150r/min.After a period of time is incubated,
The Microcystins Concentration in reaction system is detected, and is converted into degradation efficiency to embody degradable micro-capsules of the pH to the present invention
The influence of the efficiency of the composition degradation algae toxin of algae toxin.
The results are shown in Figure 5, and protein concentration can carry out Microcystin effective under conditions of 140-350mg/l
Degradation.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Sequence table
<110>He'nan University of Urban Construction
<120>A kind of bacillus of degradable Microcystin and its application
<141> 2017-11-10
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1220
<212> DNA
<213>Bacillus MCS1 (Bacillus sp. MCS1)
<400> 1
gtgagtaacg gctcacccaa ggccacgatg cgtagcgact gagaggggtg atcgtcacac 60
tgggactgag acacgtccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 120
acgaaagtct gaccgagcaa cgccgcgtga acgatgaagg cctccgggtc gtaaagttct 180
gttgttaggg aagaacaagt accagagtaa ctgctggtac cttgacggta cctaacccag 240
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 300
ggaattattg ggcgtaaagc gcgcgcaggc ggtttcttaa gtctgatgtg aaagcccacg 360
gctcaaccgt ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 420
ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 480
ctctggtctg taactgacgc tgaggcgcga aagcgtgggg agcgaacagg attagatacc 540
ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg 600
ctgcagcaaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 660
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 720
gaaccttacc aggtcttgac atccttcgct acttctagag atagaaggtt ccccttcggg 780
ggacgaagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 840
gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 900
tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 960
gacctgggct acacacgtgc tacaatggat ggtacaaagg gctgcaagac tgcgaagtca 1020
agccaatccc ataaaaccat tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag 1080
ccggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1140
cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgggg taaccgtaag 1200
gagccagccg cctaaggtga 1220
Claims (10)
1. a kind of bacillus of degradable Microcystin, which is characterized in that the bacillus protected on June 26th, 2017
China typical culture collection center is hidden in, preserving number is CCTCC NO:M2017381.
2. application of the bacillus described in claim 1 in degrading microcystic toxins.
3. application of the bacillus described in claim 1 in the composition for preparing degradable Microcystin.
A kind of 4. method for the composition for preparing degradable Microcystin, which is characterized in that including from described in claim 1
The step of bacillus extracts to obtain the composition of the degradable Microcystin.
5. according to the method described in claim 4, it is characterized by comprising the following steps：
S1：It is inoculated with and cultivates the bacillus and obtain bacillus culture, the bacillus culture is condensed into bacterium
Body concentrate；
S2：The concentration liquid is crushed, obtains bacterial cell disruption liquid；
S3：The bacterial cell disruption liquid is centrifuged, takes supernatant to get to the composition of the degradable Microcystin.
6. according to the method described in claim 5, it is characterized in that, the state in S1 before the bacillus is inoculated with is -20
When freezing state at the temperature below DEG C, before inoculated and cultured, the bacillus is activated.
7. according to the method described in claim 5, it is characterized in that, by centrifugation so bacillus culture collects bacterium in S1
Body, and the thalline is resuspended to carry out the concentration with the phosphate buffer of the 50mmo l/L of small size.
8. according to the method described in claim 5, it is characterized in that, in S2, the method by ultrasonication will crush.
9. according to the method described in any one of claim 5-8, which is characterized in that further include step S4 after S3：To described
Protein concentration in the composition of degradable Microcystin is quantified.
10. a kind of composition of degradable Microcystin, which is characterized in that by described in any one of claim 5-9
Method is prepared.
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