CN103555696B - Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA) - Google Patents
Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA) Download PDFInfo
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Abstract
The invention discloses a biosynthesis method for obtaining a high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA), belonging to the field of biotechnology application or environmental protection. The method comprises the following steps: constructing the sequences shown in SEQ ID NO.1 on pMAL-C2X to obtain pMAL-MlrA; transferring pMAL-MlrA to escherichia coli TB1 to obtain an expression bacterium of MlrA; carrying out induced expression on the expression bacterium of MlrA with isopropyl beta-D-1-thiogalactopyranoside (IPTG), collecting thalli after induced expression, crushing and purifying by using a maltose-binding protein (MBP) tag, thus obtaining the high-purity and high-efficiency MlrA. By optimizing an expression vector and expression conditions, the purity of the obtained MBP-MlrA is above 90% and the purity of MlrA after cutting off the MBP tag can be above 98%. The obtained MlrA has wide range of degradation and high degradation efficiency.
Description
Technical field
The present invention relates to biotechnology applications or field of environment protection, be specifically related to a kind of biosynthetic means obtaining high-purity efficient algal toxin degradation enzyme.
Background technology
Algae toxins (Microcystins, MCs) is a serious toxoid (Duy T.N. et al. Toxicology and risk assessment of fresh water eyanobacteria (blue-green algae) the toxic in water. of harm that blue-green algae discharges in process of growth and after death
rev. Environ. Contam. Toxicaol. 2000,163,113-186), its structure iron is as shown in Figure 1.MCs is ring-type seven peptide compounds, whole ring type polypeptide is divided into 7 parts: 1, D L-Ala; 2, R1; The different aspartic acid of 3, D-Beta-methyl; 4, R2; 5, Adda; 6, D isoglutamic acid; 7, N-methyl prednisolone L-Ala.At present existing 67 kinds of Algae toxins are found, this difference be mostly 2 in ring texture, 3,4 different caused with the amino-acid residue of 7.Modal have MC-LR and MC-RR two kinds, in the ring texture of wherein MC-LR, the amino-acid residue of 2 and 4 is respectively lysine residue (L) and arginine residues (R), and in the ring texture of MC-RR, the amino-acid residue of 2 and 4 is all arginine residues (R).MCs pollution can cause the death of water body animal, and the water drunk by Algae toxins pollutes can bring out the liver injury such as people, animal, fowl, cause liver cancer etc.
In recent years, due to the raising of various places water body eutrophication degree, blue-green alga bloom phenomenon frequently breaks out, and the blue-green algae of these growths or death can discharge a large amount of Algae toxins, and the harm caused the mankind and environment is day by day serious.The detoxification of MCs or removing toxic substances seem particularly urgent.Simultaneously due to the raising of people's living standard, the security of water body treating, validity are had higher requirement.
Mainly contained at present by the method removed or Algae toxins of degrading reaches removing toxic substances object: (1) physical chemistry edman degradation Edman: by solidifying, gac absorbs, membrane technology or UV/H
2o
2clO
2degraded Algae toxins.Although solidify, gac absorbs, membrane technology partly can reduce the content of Algae toxins in water, algal toxin degradation can not effectively be fallen by place Algae toxins can only being transferred to other, and consumption material consuming time.Although the method degradation efficiency of oxidative degradation Algae toxins is higher, low price, because the ability of killing livestock of oxygenant is strong, specificity is poor, and easy and organic pollutant reacts the material producing negative unhealthy.(2) degradation by bacteria method: this method utilize fine sand filter and containing there is the microbial film of optionally biological modeling to process water sample, this method economy, but complex operation, need often to change microbial film, and biological modeling own metabolism needs to consume nutrient, the easy stability affecting the ecosystem, and unknown secondary metabolism toxic substance may be produced, cause secondary pollution.(3) bio-enzyme degradation method: biodegradable enzyme can be degraded Algae toxins, makes it change low toxicity, nontoxic small-molecule substance into, has specificity, high efficiency and can not produce the features such as toxic substance thus receive much concern.
Bio-enzyme degradation Algae toxins removes Algae toxins to one of best practice of the pollution problem of water body effectively, safely beyond doubt at present.The reports such as Bourne D.G. in 1996: pass through
sphingomonas sp.to the degraded product analysis of MC-LR, infer that degraded MC-LR has four enzyme (MlrA at least, MlrB, MlrC, MlrD) wherein three lytic enzyme (MlrA, MlrB, the oligopeptide transmission factor (MlrD) of MlrC) and one supposition, and infer a kind of may MC-LR degradation pathway. first enzyme MlrA is a very important enzyme, be wire Algae toxins by ring-type algal toxin degradation, Algae toxins toxicity is made to reduce nearly 160 times of (Bourne D.G., et al. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. Appl. Environ. Microbiol. 1996, 62, 4086-4094.).Calendar year 2001 Bourne D.G. etc. has cloned this four genes, and has carried out sequencing and sign to these four genes.Hai Yan in 2012 etc. from
sphingopyxis sp.in cloned gene USTB-05-A, and this gene USTB-05-A to be proceeded to PGEX4T-1 be that carrier is expressed at BL21 (DE3), USTB-05-A gene with reported
sphingomonas sp.mlrA(GeneBank No. AF411068 in ACM-3962) nucleic acid similarity is 92.5%(Yan H., et al, Characterization of the first step involved in enzymatic pathway for microcystin-RR biodegraded by
sphingopyxis sp.uSTB-05. Chemosphere. 2012,87,12-18).But these experiments are what to carry out under the condition of mixed enzyme solution at present, and do not obtain pure enzyme, the expression amount of enzyme and expression activity are not high, have impact on the further application of algal toxin degradation enzyme.
Therefore how obtaining high-purity efficient algal toxin degradation enzyme, is that our applying biological enzyme removes Algae toxins to water pollution important prerequisite and a matter of utmost importance effectively, safely.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art and deficiency, a kind of biosynthetic means obtaining high-purity efficient algal toxin degradation enzyme (MlrA) is provided.
Object of the present invention is achieved through the following technical solutions:
A kind of biosynthetic means obtaining high-purity efficient algal toxin degradation enzyme (MlrA), comprises the steps:
(1) the MlrA sequence construct such as shown in SEQ ID NO.1 to prokaryotic expression carrier pMAL-C2X will obtain the carrier pMAL-MlrA expressing MlrA.
(2) pMAL-MlrA is transformed into the expression bacterium obtaining MlrA in intestinal bacteria TB1.
(3) by the expression bacterium IPTG abduction delivering of MlrA, collect the thalline after abduction delivering, the expressing fusion protein label on pMAL-C2X (maltose binding protein MBP, relative molecular weight 42500) purifying is utilized to obtain high-purity efficient algal toxin degradation enzyme after fragmentation.
Carrier pMAL-MlrA described in step (1) is preferably by the preparation of the method for following steps: be loaded in cloning vector pUC19 by the such as MlrA shown in SEQ ID NO.1 by synthesis, obtain pUC19-MlrA; Take pUC19-MlrA as template, with MlrA upstream primer and downstream primer amplification MlrA; By the restriction enzyme site BamH I on upstream and downstream primer and Hind III, MlrA is building up on pMAL-C2X carrier, obtains the carrier pMAL-MlrA expressing MlrA;
Wherein, MlrA upstream primer is 5 '-GCGGATCCATGCGGGAGTTTGTCAAACAG-3 ', MlrA downstream primer is 5 '-CGCAAGCTTTCACGCGTTCGCGCCGGACTT-3 '.
Inductive condition described in step (3) is preferably: before induction, bacterium liquid OD600 is 1.0, IPTG concentration is 0.5mM, and abduction delivering temperature is 22 DEG C, and induction time is 8 hours.
The method of the purifying described in step (3) preferably includes following steps: with NaOH solution, the column balance buffering liquid washing MBP post of redistilled water, 0.5M, balance purification column (regeneration of MBP post); By resuspended for the thalline column balance buffering liquid after the abduction delivering of collection, ultrasonication, centrifuging and taking supernatant, with the MBP resin-bonded after balance; Combining mixed solution is poured in void column, first rinses with column balance buffering liquid and foreign protein is split away off from resin, then collect with elution buffer wash-out the algal toxin degradation enzyme MlrA that target protein obtains purifying; Wherein, column balance buffering liquid component is: 36mM KH
2pO
4, 18mM K
2hPO
4, 200mM KCl; Elution buffer fluid component is: 0.36g maltose, column balance buffering liquid 100mL.
Compared with prior art, the present invention has the following advantages and effect: by optimization expression carrier and expression condition, the algal toxin degradation enzyme MBP-MlrA purity obtained reaches more than 90%, algal toxin degradation enzyme MlrA purity after excision MBP label can reach more than 98%, and verified that this enzyme can be degraded modal two kinds of Algae toxins MC-LR and MC-RR simultaneously, degradation efficiency is 41.2 μ g/min/mg.pr and 47.3 μ g/min/mg.pr respectively.And the MlrA of bibliographical information is mostly crude extract at present, only degrade MC-RR or MC-LR, and the proteolytic degradation MC-RR efficiency of the USTB-05-A genetic expression of latest report is also only 7.6 μ g/min/mg.pr(Yan H. et al., 2012).
Accompanying drawing explanation
Fig. 1 is Algae toxins structure iron; MCs is ring-type seven peptide compounds, whole ring type polypeptide is divided into 7 parts: 1, D L-Ala; 2, R1; The different aspartic acid of 3, D-Beta-methyl; 4, R2; 5, Adda; 6, D isoglutamic acid; 7, N-methyl prednisolone L-Ala; In MC-LR, R1, R2 are leucine (Leu) and arginine (Arg) respectively; In MC-RR, R1, R2 are arginine (Arg); In MC-YR, R1, R2 are tyrosine (Tyr) and arginine (Arg) respectively.
Fig. 2 is that PCR screens positive monoclonal agarose gel electrophoresis figure; M:DL2000,1 ~ 4: positive monoclonal.
Fig. 3 is the SDS-PAGE figure of different bacterial concentration condition before induction; M is Marker, 1 and 2 is that OD600 reaches 0.6 not inducing and inducing respectively, 3 and 4 is that OD600 reaches 1.0 not inducing and inducing respectively, 5 and 6 is that OD600 reaches 1.4 not inducing and inducing respectively, in this experiment, inductor IPTG concentration is 0.5mM, abduction delivering temperature is 22 DEG C, and induction time is 8 hours.
Fig. 4 is the SDS-PAGE figure of different inductor IPTG concentration conditions; M:Marker, 1: do not induce, 2 ~ 6: inductor IPTG concentration is 0.05mM, 0.5mM, 1.0mM, 5mM, 10mM respectively; Bacterial concentration OD600=1.0 before induction in this experiment, abduction delivering temperature is 22 DEG C, and induction time is 8 hours.
Fig. 5 is the SDS-PAGE figure of different inducing temperature condition; M is Marker, 1 and 2 precipitation and the supernatants being respectively abduction delivering at 16 DEG C, 3 and 4 precipitation and the supernatants being respectively abduction delivering at 22 DEG C, and 5 and 6 is precipitation and the supernatant of abduction delivering at 37 DEG C respectively; Bacterial concentration OD600=1.0 before induction in this experiment, inductor IPTG concentration is 0.5mM, and induction time is 8 hours.
Fig. 6 is the SDS-PAGE figure of different induction time condition under front bacterial concentration OD600=1.0 of induction are induced at 0.5mM IPTG and 22 DEG C; M is Marker, and 1 ~ 4 induction time is 0h, 4h, 6h, 8h respectively.
Fig. 7 expresses under optimal conditions and the SDS-PAGE of purifying algal toxin degradation enzyme MlrA figure; M, Marker; 1, do not induce bacterium liquid; 2, induction bacterium liquid; 3, the centrifugal precipitation obtained after smudge cells; 4, the centrifugal supernatant obtained after smudge cells; 5, stream wears liquid (mixed liquid of protein does not have bound fraction through column); 6, wash-out foreign protein; 7, the MBP-MlrA after purifying; 8, the MlrA after excision label B MP.
Fig. 8 is MC-RR canonical plotting.
Fig. 9 is MC-LR canonical plotting.
Figure 10 is the algal toxin degradation enzyme liberating MC-RR active HPLC detected result figure of 30 minutes.
Figure 11 is the algal toxin degradation enzyme liberating MC-LR active HPLC detected result figure of 30 minutes.
Figure 12 is the result figure of the product of mass spectroscopy MC-LR, MC-RR and algal toxin degradation enzyme liberating MC-LR, MC-RR; A:MC-RR, MW(molecular weight)=1037.7; B: the product of algal toxin degradation enzyme liberating MC-RR, MW=1055.9; C:MC-LR, MW=994.6; D: the product of algal toxin degradation enzyme liberating MC-LR, MW=1013.6.
Figure 13 is the mechanism figure that mass spectrum infers MlrA degraded MC-LR and MC-RR; The mechanism of degradation of A:MC-LR, the mechanism of degradation of B:MC-RR.
Embodiment
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
the structure of embodiment 1 algal toxin degradation expression of enzymes carrier pMAL-C2X-MlrA
By MlrA(nucleotide sequence as shown in SEQ ID NO.1) be loaded in cloning vector pUC19 by synthesis, obtain pUC19-MlrA.
(1) extraction of plasmid
Get 2 and 20mL LB(Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L, penbritin 30mg/L be housed) Erlenmeyer flask of substratum, single bacterium colony containing pUC19-MlrA, pMAL-C2X plasmid is added in substratum respectively, 37 DEG C, incubated overnight under 220rpm, extract plasmid according to SDS cracking process.Carried out mark after plasmid extraction completes to put into 4 DEG C of refrigerators and wait for that next step uses.
(2) double digestion of pcr amplification MlrA, MlrA and pMAL-C2X
Pcr amplification reaction system: plasmid (pUC19-MlrA) 1 μ L, 10 × buffer 5 μ L, dNTPs 4 μ L, MlrA upstream primer 1 μ L, MlrA downstream primer 1 μ L, Taq DNA polymerase 1 μ L, add ddH
2o to 50 μ L.MlrA upstream primer: 5 '-GC
gGATCCaTGCGGGAGTTTGTCAAACAG-3 ', MlrA downstream primer: 5 '-CGC
aAGCTTtCAC
GCGTTCGCGCCGGACTT-3 ', underlined sequences is BamH I and Hind III restriction enzyme site.
Pcr amplification condition: 94 DEG C of denaturation 5min; 94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations; Finally extend 72 DEG C of 5min.
PCR primer MlrA is detected correctly by agarose gel electrophoresis, carries out glue recovery and carry out double digestion with BamH I and Hind III.PMAL-C2X carrier (the expressing fusion protein label of this carrier is maltose binding protein MBP, relative molecular weight 42500) also carries out double digestion with BamH I and Hind III.Digestion products carries out glue and reclaims for connecting.
(3) connect and transform
Add respectively in EP pipe enzyme cut after carrier pMAL-C2X 2 μ L, 10 × T4 DNA ligase damping fluid 1 μ L, T4 DNA ligase 1 μ L, H
2o 5 μ L, enzyme cut after goal gene MlrA 1 μ L mix, the amount alterable of carrier and goal gene, puts into the constant water bath box of 16 DEG C by EP pipe, water-bath 16h.Carrier and goal gene can connect together by this step.
Obtained competence DH5 α is taken out from refrigerator and is put in freeze thawing on ice, add 10 μ L and connect product, EP pipe is refitted in 30min in ice, then puts in the water-bath of 42 DEG C, place 90s, then put into ice, leave standstill 5min.Often pipe adds 600 μ L LB substratum, in shaking table 37 DEG C, 180rpm cultivates 60 ~ 90min; Then 4000rpm is centrifugal, gets 600 μ L supernatants, precipitation is mixed gently, is coated on the LB flat board containing penbritin 30mg/L, in 37 DEG C of thermostat containers, cultivate 13 ~ 16h.
(4) mono-clonal detects
By single bacterium colony that upper step flat board grows, to activate in containing the flask of penbritin 30mg/L20mL LB substratum 37 DEG C, 220rpm cultivates 16h.Get bacterium liquid 3 μ L and carry out PCR checking (Fig. 2), get plasmid (pUC19-MlrA) 3 μ L and water 3 μ L respectively to carry out PCR and do the positive and negative control, PCR primer agarose gel electrophoresis is detected, if the band that bacterium liquid amplifies is identical with the band molecular weight that plasmid pUC19-MlrA amplifies, then preliminary proof successful connection.
Reactivated by bacterium liquid corresponding to correct band, protect bacterium and send to order-checking, the MlrA fragment length that the display of sequencing sequence result is loaded into is 1008bp, and compared by BLAST in NCBI, comparison unanimously shows that carrier pMAL-C2X-MlrA successfully constructs.
the expression and purification of embodiment 2 algal toxin degradation enzyme
(1) optimization of inductive condition
Bacterial concentration, inductor IPTG concentration, abduction delivering temperature, induction time before induction are optimized
Extract the pMAL-C2X-MlrA plasmid in bacillus coli DH 5 alpha, the plasmid of extraction is proceeded in TB1 competence.Expression bacterium containing goal gene to be activated in the flask of the 20mL LB substratum containing penbritin 30mg/L 37 DEG C, 220rpm cultivates 16h, get the bacterium liquid after activation by 2.5% inoculum size be inoculated in 500mL LB substratum, select different inductive conditions to carry out abduction delivering.Thalline is collected, by SDS-PAGE testing goal protein expression situation, to determine optimum inductive condition after abduction delivering.Result as illustrated in figures 3-6, finally determines that optimum inductive condition is: before induction, bacterium liquid OD600 is 1.0, and inductor IPTG concentration is 0.5mM, and abduction delivering temperature is 22 DEG C, and induction time is 8 hours.
(2) preparation of inducible strain
Expression bacterium containing goal gene to be activated in the flask of the 20mL LB substratum containing penbritin 30mg/L 37 DEG C, 220rpm cultivates 16h, get the bacterium liquid after activation by 2.5% inoculum size be inoculated in 500mL LB substratum, when cultivating OD600 1.0, add IPTG to final concentration 0.5mM, 22 DEG C, 220rpm induces 8 hours, with the bacterium after high speed freezing centrifuge (J-26XP, Beckman) 12000rpm, 4 DEG C of collected by centrifugation inductions.
(3) regeneration of MBP post
First 5 column volumes are washed with second distillation, 3 column volumes are washed afterwards by the NaOH solution of 0.5M, fill NaOH and soak 30min, again with NaOH solution washing 1 ~ 2 column volume of 0.5M, use second distillation water washing 10 column volumes again, until no longer include NaOH solution in pillar, then use column balance buffering liquid (36mM KH
2pO
4, 18mM K
2hPO
4, 200mM KCl) and wash 5 column volumes, balance purification column.
(4) protein purification
Purifying protein damping fluid used:
Column balance buffering liquid: 36mM KH
2pO
4, 18mM K
2hPO
4, 200mM KCl;
Elution buffer: 0.36g maltose, column balance buffering liquid 100mL.
Bacterial precipitation after induction is placed on freeze thawing on ice, with 30mL column balance buffering liquid suspended bacterial, ultrasonication (runs 3 seconds, stop 3 seconds, 180 circulations) to solution becomes translucent, then use high speed freezing centrifuge (J-26XP, Beckman) 12000rpm, 4 DEG C of centrifugal 40min, get supernatant with getting the membrane filtration of supernatant with 0.45 μm.
The MBP post resin 4 DEG C regenerated by the supernatant filtered is in conjunction with 2h, after 2h, the mixed solution of thick enzyme and resin is poured in void column, all adding after in pillar until it makes foreign protein split away off from resin with column balance buffering liquid flushing resin, check the amount of foreign protein with Xylene Brilliant Cyanine G, foreign protein wash clean is substantially described after Xylene Brilliant Cyanine G does not develop the color.Finally in resin, add 5mL elution buffer, then collect target protein with a clean evaporating pipe and check target protein with Xylene Brilliant Cyanine G equally.At 4 DEG C, 4000rpm is centrifugal, and protein concentrate, until protein concentration reaches more than 1mg/mL, is deposited into-20 DEG C of preservations with glycerine 1:1 mixed protein.The SDS-PAGE result of purifying protein is obtained as shown in Figure 7 under optimal conditions, target protein MBP-MlrA purity is more than 90%, by finding out with contrasting of albumen Marker, the monomer molecule amount of albumen is about 66 KDa, MBP(maltose binding protein) label protein size is about 40 KDa, so the monomer molecule amount removing label protein algal toxin degradation enzyme is about 36 KDa, what calculate with the amino acid (sequence is as shown in SEQ ID NO.2) according to algal toxin degradation enzyme enzyme meets substantially, MlrA purity of protein after excision MBP label is more than 98%.
the Activity determination of embodiment 3 algal toxin degradation enzyme
(1) the cleaning balance of chromatographic column
First open the aqueous solution of high performance liquid chromatograph (Agilent 1200 LC) containing methyl alcohol from 100% ~ 2% gradient elution chromatography post 2 times, finally use 60% methanol wash column to baseline, treat that baseline prepares sample after walking flat 20min.
(2) detection of sample
Sample message
Sample-loading buffer is 0.05mol/L phosphate buffered saline buffer, is adjusted to pH=7.0; Algae toxins MC-LR, MC-RR standard concentration is 10 μ g/mL, with methanol dilution to 5.0 μ g/mL, 1.0 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL; Methyl alcohol is HPLC chromatographically pure Fisher lab analysis reagent 4L; Water is redistilled water, and all samples all needs the membrane filtration with 0.22 μm, and removes the bubble dissolved the inside by ultrasonic wave.
Testing conditions
Sample size is 10 μ L, and moving phase is 60% methyl alcohol, and column temperature is 25 DEG C, and flow velocity is 0.8mL/min, and determined wavelength is 238nm.Instrument is HPLC(Agilent 1200 LC), pillar is Eclipse XDB-C18 chromatographic column (4.6mm × 15mm).
Examination criteria product 238nm place absorption peak area, formulates the typical curve of Algae toxins standard substance, calculates the relation (Fig. 8, Fig. 9) of peak area and sample concentration.
Sample detection:
1) get 200 μ L phosphate buffered saline buffers in the liquid phase bottle of 1mL, sample introduction once does control group, sees that whether baseline is steady.
2) 190 μ L phosphate buffered saline buffers, 10 μ L(10 μ g/mL are got) Algae toxins is in the liquid phase bottle of 1mL, and sample introduction does Substrate controls twice, sees that whether baseline is steady; The appearance time of record substrate and peak area.
3) get 182 μ L phosphate buffer solns, 10 μ L(10 μ g/mL) Algae toxins, 8 μ L(0.06mg/mL) the algal toxin degradation enzyme of above-mentioned preparation, mix.Measure the change of Algae toxins peak area after putting into 30 DEG C of water-bath timing 10min, whether difference detected peaks area change again after 30min, 50min, to determine the activity of algal toxin degradation enzyme, and degrade completely.As shown in Figure 10 and Figure 11, the enzyme reaction time is 30min to result.The algal toxin degradation enzyme liberating efficiency of preparation is 41.2 μ g/min/mg.pr and 47.3 μ g/min/mg.pr respectively.
(3) qualification of degraded product
MC-LR, the MC-RR of algal toxin degradation enzyme liberating with above-mentioned preparation are carried out mass spectrometric detection, detects figure as (Figure 12), according to the molecular weight of degraded product, infer the mechanism of MlrA degraded MC-LR, MC-RR as shown in figure 13.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. obtain a biosynthetic means for high-purity efficient algal toxin degradation enzyme, it is characterized in that comprising the steps:
(1) the MlrA sequence construct such as shown in SEQ ID NO.1 to prokaryotic expression carrier pMAL-C2X will obtain the carrier pMAL-MlrA expressing MlrA;
(2) pMAL-MlrA is transformed into the expression bacterium obtaining MlrA in intestinal bacteria TB1;
(3) by the expression bacterium IPTG abduction delivering of MlrA, collect the thalline after abduction delivering, after fragmentation, utilize the expressing fusion protein tag purification on pMAL-C2X to obtain high-purity efficient algal toxin degradation enzyme;
The method of the purifying described in step (3) comprises the steps: NaOH solution, column balance buffering liquid washing MBP post with redistilled water, 0.5M, balance purification column; By resuspended for the thalline column balance buffering liquid after the abduction delivering of collection, ultrasonication, centrifuging and taking supernatant, with the MBP resin-bonded after balance; Combining mixed solution is poured in void column, first rinses with column balance buffering liquid and foreign protein is split away off from resin, then collect with elution buffer wash-out the algal toxin degradation enzyme MlrA that target protein obtains purifying; Wherein, column balance buffering liquid component is: 36mM KH
2pO
4, 18mM K
2hPO
4, 200mM KCl; Elution buffer fluid component is: 0.36g maltose, column balance buffering liquid 100mL.
2. the biosynthetic means of the high-purity efficient algal toxin degradation enzyme of acquisition according to claim 1, is characterized in that:
The preparation of the carrier pMAL-MlrA method as follows described in step (1): the such as MlrA shown in SEQ ID NO.1 is loaded in cloning vector pUC19 by synthesis, obtains pUC19-MlrA; Take pUC19-MlrA as template, with MlrA upstream primer and downstream primer amplification MlrA; By the restriction enzyme site BamH I on upstream and downstream primer and Hind III, MlrA is building up on pMAL-C2X carrier, obtains the carrier pMAL-MlrA expressing MlrA;
Wherein, MlrA upstream primer is 5 '-GCGGATCCATGCGGGAGTTTGTCAAACAG-3 ', MlrA downstream primer is 5 '-CGCAAGCTTTCACGCGTTCGCGCCGGACTT-3 '.
3. the biosynthetic means of the high-purity efficient algal toxin degradation enzyme of acquisition according to claim 1, is characterized in that:
Inductive condition described in step (3) is: before induction, bacterium liquid OD600 is 1.0, IPTG concentration is 0.5mM, and abduction delivering temperature is 22 DEG C, and induction time is 8 hours.
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| CN104195129A (en) * | 2014-07-28 | 2014-12-10 | 华中师范大学 | Immobilized algal toxin degrading enzyme as well as preparation method and application thereof |
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