CN102796714B - Phosphotriesterase mutant as well as preparation method and application thereof - Google Patents
Phosphotriesterase mutant as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a phosphotriesterase mutant as well as a preparation method and application of the phosphotriesterase mutant. According to the invention, a mutant with phosphotriesterase activity markedly improved is finally obtained by starting from a phosphotriesterase gene which is from Geobacillus kaustophilus HTA426 of thermophilic bacteria and then carrying out rounds of mutation and screening through an error-prone PCR (Polymerase Chain Reaction) method; and the mutations involved in amino acid sequence of the mutant are Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys and Gly273Asp. Compared with common organic phosphorous insecticides, the wild type specific activity of the mutant is markedly improved and the mutant has wide application prospect in the field of biodegradation of organic phosphorous poisions.
Description
Technical field
The present invention relates to biotechnology, be specifically related to a kind of phosphotriester enzyme mutant and its preparation method and application.
Background technology
Phosphoric triesterase (Phosphotriesterase, EC 3.1.8.1) can be hydrolyzed the organo phosphorous compounds of wide spectrum, the latter is widely used in agricultural insecticide and chemical warfare nerve poison, phosphoric triesterase can catalysis fracture organo phosphorous compounds phosphide key and make its detoxification.Be shown below:
Organo phosphorous compounds has extremely strong toxic side effect to people and domestic animal.Some relatively common are machine phosphorus compound and toxicity thereof in table 1.So far have to exceed 100 kinds of organic phosphorous insecticides and be widely used all over the world, account for 38% of sterilant kind.Only be used for agriculture production at the annual nearly 50,000 tons of organic phosphorous insecticides of the U.S., except this approximately produces 400,000 liters of Coumaphos (coumaphos) waste liquid by " ox flea is eradicated plan " every year; And some European Union member countries produce millions of diazinon (diazinon) that rise for cleaning wool liquid medicine.Due to excessive and continuous use, many soils and water resources have been suffered serious pollution, human health has been formed to grave danger (Singh, B.K. Organophosphorus-degrading bacteria:ecology and industrial applications. Nat. Rev. Microbiol. 2009,7 (2): 156-164.).Probably there are every year in the world three million peoples because organophosphorus is taken in poisoning, wherein approximately 200,000 people's death (Karalliedde, L., Senanayake, N., Organophosphorus insecticide poisoning. J. Int. Fed. Clin. Chem. 1999,11 (2): 4-9.).On the other hand, be about 200,000 tons for the chemical warfare nerve poison of military reserve in the world, before 2023, needed whole destructions according to international these chemical weapons of chemical weapons treaty.Therefore phosphoric triesterase has wide application potential in fields such as the degraded of organophosphorus pollutent, scrubbing, biosensor, medicine research and development.
The organo phosphorous compounds that table 1 is several frequently seen and toxicity thereof
Orthogenesis is in laboratory, to simulate Darwin's natural evolution process, for the encoding gene of a certain protein, by the gene of improved induced-mutation technique transformation enzyme, builds sudden change library, then, according to specific transformation object, screens valuable non-natural enzyme.Orthogenesis technology obtains huge success in enzymatic property transformation field, mainly concentrate on the catalytic reaction activity that improves enzyme, improve the substrate specificity of enzyme, improve thermostability, change the aspect (Turner such as enantio-selectivity and the tolerance of raising to organic solvent of enzyme, N.J. Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol. 2003, 21 (11): 474-478. Johannes, T.W., and Zhao, H. Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol. 2006, 9 (3): 261-267).
Summary of the invention
Geobacillus kaustophilus HTA426 bacterial strain is that the people such as Takami separate and obtain in the mud sample in Mariana Islands seabed, belong to gram positive bacterium, be kept at Japanese microbial preservation center (Japan Collection of Microorganisms, http://www.jcm.riken.jp/), preserve and be numbered JCM 12893.It can exceed 70oC, growth (Takami under the extreme condition of pH9.5-10, H., Inoue, A., Fuji, F., and Horikoshi, K. Microflora in the deepest sea mud of the Mariana Trench. FEMS Microbial. Lett. 1997,152 (2): 279-285).This indicates that it self albumen will have excellent biological stability, and this strain gene group sequence is announced (http://www.ncbi.nlm.nih.gov/genome/1659) at state-run biotechnology information center of U.S. genome database.From genome sequence, it contains phosphotriester enzyme coding gene (GK1506).We utilize polymerase chain reaction (Polymerization chain reaction, PCR) increased from G. kaustophilus HTA426 genome GK1506 gene be inserted into pET28a expression plasmid of technology, recombinant plasmid is transferred in escherichia coli host, obtain a strain engineering bacteria, called after pET28a-GkaP (WT).This project bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 15th, 2011, be called for short CGMCC, address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica.Deposit number is CGMCC No.5598.Its Classification And Nomenclature is: colon bacillus, latin name: Escherichia coli.
Technical scheme of the present invention is: the phosphoric triesterase body variant that chamber evolution technology structure vigor improves by experiment.The phosphoric triesterase gene (Genbank accession number 3183579) that origin comes from G. kaustophilus HTA426 carries out molecular evolution, taking the expression plasmid that contains phosphoric triesterase gene that builds as template, the method of fortune fallibility PCR, through too much sudden change and the screening of wheel, finally obtain a phosphotriester enzyme mutant that organic phosphorus degrading vigor significantly improves.Adopt " amino acid of original amino acid-position-sudden change " to represent the aminoacid replacement of mutant, the amino acid mutation containing in described mutant is: Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys, Gly273Asp.The colibacillus engineering strain (called after pET28a-GkaP (8M)) of expressing this mutant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 15th, 2011, be called for short CGMCC, address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica.Deposit number is CGMCC No.5597.Its Classification And Nomenclature is: colon bacillus, latin name: Escherichia coli.
G. the original amino acid of the phosphoric triesterase of kaustophilus HTA426 is as shown in SEQ ID NO:1; The aminoacid sequence of mutant is as shown in SEQ ID NO:2, and the amino acid after 8 sudden changes marks by black box; The nucleotide sequence of coding G. kaustophilus HTA426 phosphoric triesterase wild type gene is as shown in SEQ ID NO:3; Encode the nucleotide sequence of described mutant gene as shown in SEQ ID NO:4, and underscore represents the Nucleotide after sudden change.
Be pET28a for the expression vector of expressing G. kaustophilus HTA426 phosphoric triesterase and mutant thereof; The microbial host cell transforming for described expression vector is e. coli bl21 (DE3) Codon Plus.
The present invention, by protein engineering, has successfully obtained the mutant that organophosphorus hydrolysis vigor significantly improves.Optimum mutant is compared with wild-type enzyme, and it is for the catalytic efficiency (k of sterilant paraoxon
cat/ K
m) improve 232 times; 221,313,497 and 68 times are improved respectively for paraoxon, thiophos, diazinon, four kinds of common organic phosphorous insecticides of chlorpyrifos than vigor.
Brief description of the drawings
Fig. 1 is the order-checking peak figure of phosphotriester enzyme mutant 8M (Phe28Ile/Tyr99Leu/Thr171Ser/Phe228Leu/ Asn269Ser/Val270Gly/Trp271Cys/Gly273Asp), and in figure, arrow institute labeling position is the Nucleotide of introducing sudden change.
Fig. 2 is that wild-type GkaP and mutant are to four kinds of common organic phosphorous insecticide vigours.
Fig. 3 is the reaction optimum temperuture of wild-type GkaP and mutant.
Fig. 4 is the reaction optimal pH of wild-type GkaP and mutant.
Embodiment
Method used in following embodiment, be if no special instructions ordinary method, concrete steps can be referring to: " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, Dsvid W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
Embodiment 1: the clone of wild-type phosphoric triesterase gene
(1) cultivation of thermophilic bacterium Geobacillus kaustophilus HTA426 and the extraction of genomic dna thereof
The cultural method providing according to Japanese microbial preservation center is recovered and cultivates, first prepare Rehydration fluid 7:5g/L peptone (peptone), 3g/L beef extract (beef extract), 10g/L NaCl, use deionized water constant volume, regulate pH to 7.0, autoclave sterilization.
The cell lyophilized powder of Geobacillus kaustophilus HTA426 adds 0.5ml rehydration fluid, after dissolving, is transferred in the test tube that 5ml LB substratum is housed, and 60 DEG C, 180rpm shaking culture 24 hours.Then being transferred in the triangular flask that 100ml LB substratum is housed 60 DEG C cultivates 48 hours.
Get the bacterial cultures of 5 ml and collect thalline, by the resuspended precipitation of 25mmol/L Tris-HCl damping fluid (pH8.0, containing 50mmol/L glucose, 10mmol/L EDTA) of 200 μ L, add the 50mg/ml N,O-Diacetylmuramidase of 50 μ L, 4 DEG C digest 1 hour; Add the SDS solution (final concentration is 2% W/V) of 125 μ L to react 10 minutes; Add isopyknic phenol: chloroform: primary isoamyl alcohol, mix, centrifugal 5 minutes, supernatant liquor is transferred in another centrifuge tube; In precipitation, add the dehydrated alcohol of 2 times of volumes, precipitation thallus DNA, centrifugal 5 minutes of 12000rpm, goes after supernatant, with 70% washing with alcohol DNA; After centrifugal, remove supernatant, again dissolve with the deionized water of sterilizing, gained genomic dna solution is put in-20 DEG C of refrigerators for subsequent use.
(2) clone of phosphoric triesterase gene and construction of recombinant plasmid
In Geobacillus kaustophilus HTA426 genome, contain a phosphotriester enzyme coding gene (GK1506).GK1506 gene is to be increased and obtain from the genomic dna of above-mentioned bacterial strains by PCR method.Two primers are to design according to many restriction enzyme sites of the sequence of GK1506 gene and expression plasmid, entrust Shanghai Sheng Gong bio-engineering corporation synthetic.
Upstream primer: 5'-GCGC
gGATCCaTGGCGGAGATGGTAGAAACGGTAT-3', line part is BamH I site;
Downstream primer: 5'-GATC
aAGCTTgTCAAGCCGAGAACAGCGCCGCCGGAT-3', line part is HindIII site.
The restriction enzyme site that two primers are set and BamH I and the HindIII of expression plasmid pET28a match, and are suitable at E. coli.
PCR reaction system: contain 1 μ l archaeal dna polymerase (the super fidelity dna polysaccharase of the Phusion of NEB company) in 50 μ l reaction systems, 5 μ l 10 × damping fluids, 2 μ l dNTP mixtures (every kind of nucleotide concentration 2.5 mM), 2 μ l Geobacillus kaustophilus HTA426 genomic dnas, (20pmol/ μ l) for 1 μ l upstream primer, 1 μ l downstream primer (20pmol/ μ l), 38 μ l ultrapure waters.PCR reaction conditions is: first 95 DEG C of denaturation 5min; Each circulation comprises 95 DEG C of sex change 40s, 58 DEG C of annealing 40s, and 72 DEG C are extended 90s, totally 30 circulations; Last 72 DEG C are extended in.Reaction finishes rear use 1% agarose gel electrophoresis and detects PCR product.Molecular weight is consistent with (981bp) of expection.Use purification kit to carry out purifying to PCR product.
The PCR product of purifying is cut to (NEB company) with restriction enzyme BamH I and HindIII enzyme, at 37 DEG C, hatch 3 hours.Enzyme cuts complete, electrophoresis on the sepharose of 1.0 %, and application DNA gel test kit reclaims the DNA fragmentation after enzyme is cut.
Apply same enzymic digestion pET28a plasmid, and then (every 50 μ l systems add 1 μ l to process plasmid with alkaline phosphatase (Fermentas company), 37 DEG C of insulation 1h), in 0.8% sepharose, detect linear carrier and reclaim purifying.Then the goal gene fragment of processing and linear carrier fragment are utilized T4 DNA ligase (NEB company) to be connected.Connection product is proceeded in e. coli bl21 (DE3) Codon Plus, use containing the LB solid medium of 50 μ g/ml kantlex and carry out positive colony screening.Random picking mono-clonal checks order, and result is consistent with report, has proved to obtain the recombinant plasmid that contains phosphoric triesterase gene, called after pET28a-GkaP.
Embodiment 2: utilize the method for fallibility PCR to build random mutation storehouse
Utilize the method for fallibility PCR to introduce random nucleotide sudden change in G. kaustophilus HTA426 phosphoric triesterase gene.The primer is with to clone phosphoric triesterase gene the primer in embodiment 1 identical.Error-prone PCR systems is as follows: in 100 μ l reaction systems, containing 2 μ l Taq polysaccharases (TaKaRa company), 10 μ l 10 × damping fluids are not (containing Mg
2+), 8 μ l dCTP (10mM), 8 μ l dTTP (10mM), 8 μ l dNTP mixtures (every kind of nucleotide concentration 2.5 mM), 24 μ l MgCl
2(25mM), 2 μ l MnCl
2(10mM), 1 μ l carries the pET28a recombinant plasmid of phosphoric triesterase gene, 2 μ l upstream primers (20pmol/ μ l), 2 μ l downstream primers (20pmol/ μ l), 33 μ l ultrapure waters.Fallibility PCR reaction conditions is identical with the condition described in embodiment 1.Reaction finishes rear use 1% agarose gel electrophoresis and detects fallibility PCR product.
After fallibility pcr amplification product is purified, restriction enzyme BamH I and HindIII digest fallibility PCR product and expression plasmid pET28a respectively, connect.Connection product electric shocking method after purifying is transformed in e. coli bl21 (DE3) Codon Plus competent cell.Coat in the LB resistant panel that contains 50ug/ml kantlex, cultivate 12 hours at 37 DEG C, the whole transformants of gained are random mutation storehouse.Random 10 transformants of picking check order, existence and the mutation rate of checking coding mutation.Transformant, through order-checking, determines that the mutation rate that fallibility PCR introduces is 1-5 base/gene.
Embodiment 3: the screening of mutant
From sterilizing toothpick picking clone mutation library, be inoculated into respectively in the 96 porocyte culture plates that contain 50ug/ml kantlex LB liquid nutrient medium containing 200 μ l in every hole, every hole is corresponding to each specific transformant.Meanwhile, in 96 porocyte culture plates, inoculate wild-type clone and empty bacterium, respectively as positive control and negative control.37 DEG C, 160 rpm, shaking table is cultivated approximately 16 h, makes bacterial growth reach plateau.It is 20% that every hole adds the aseptic glycerine of 50% v/v to glycerine final concentration, frozen in-80 DEG C.
From the bacterium hole of former bacterium plate, taking out 5 μ l bacterium liquid goes to and adds in advance 150 μ l LB liquid nutrient mediums (to include 50ug/ml kantlex, 1mM Co
2+) the corresponding aperture of 96 well culture plates in.Copy board is in 37 DEG C, and the careful jolting of 180 rpm is cultured to OD
600for 0.2-0.30 left and right.It is that 1 mM inducible protein is expressed that every hole adds isopropylthio β-D-galactoside (IPTG) to final concentration, and then in 37 DEG C, 150 rpm joltings are cultured to OD
600for 0.9-1.0 left and right, measure OD
600.Ductor is put into refrigerated centrifuge, in 3000 rpm, 4 DEG C, centrifugal 30 min, supernatant discarded.Thalline, in-40 DEG C of multigelations three times, then adds 50 mM in every hole, and the 2-cyclohexylamino ethyl sulfonic acid damping fluid of pH 9.0 is (containing 0.2 mM Co
2+) 200 μ l, suspension cell.In 3000 rpm, 4 DEG C, centrifugal 40 min, shift in 100 μ l supernatant to 96 hole enzyme plate corresponding apertures, are crude enzyme liquid.
In the 96 hole enzyme plates that contain 100 μ l crude enzyme liquids, add 100 μ l 50 mM, the 2-cyclohexylamino ethyl sulfonic acid damping fluid (containing the substrate paraoxon of 0.5 mM) of pH 9.0 mixes, 37 DEG C of reaction 10 min.After finishing, reaction in microplate reader, measures immediately OD
405and save data.Calculate each clone OD
405/ OD
600numerical value, compare with the ratio of positive control, result is chosen higher than the clone of positive control.By continuous 4 sudden change of taking turns and screenings, the clone's quantity that amounts to screening is about 10,000.Finally obtain optimum mutant.What in the aminoacid sequence of this mutant, relate to sports Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys, Gly273Asp, and the nucleotide sequence order-checking peak figure of its coding is shown in Fig. 1.
Embodiment 4: the expression of phosphoric triesterase wild-type and mutant thereof and purifying
The intestinal bacteria of the pET28a recombinant plasmid that contains phosphoric triesterase (wild-type or mutant) gene are inoculated into 37 DEG C of shaking culture in the LB substratum that 5 ml contain 50ug/ml kantlex to spend the night; Be inoculated into next day in the fresh culture of the identical resistance of 100mL, 37 DEG C are continued to cultivate 4 h.The seed liquor of tentatively amplifying is accessed with 1% ratio in the LB substratum of 2 L (containing 1mM Co
2+), cultivate (37 DEG C, 180rpm) in air table concussion.Treat that thalli growth is to OD
600it is 0.6 ~ 0.8 o'clock, adding isopropylthio β-D-galactoside (IPTG) to the final concentration of 100mM is 1mM, reduce culture temperature (22 DEG C), thalline is induced and made it produce a large amount of target proteins, avoid producing the inclusion body of enzyme simultaneously, cultivate and gather in the crops thalline after 16 hours.
(pH8.0, containing 0.2mM Co to add the 50mM Tris-HCl damping fluid of 5-10 times of volume
2+) resuspended thalline, ultrasonication 10 minutes; By broken liquid, in 60 DEG C of heat treated 30 minutes, the centrifugal intestinal bacteria foreign protein (12000rpm, 30min) of removing sex change, collected supernatant and obtains crude enzyme liquid.
Because it is histidine-tagged that expressed target protein N-end contains, application of nickel affinity column carries out purifying to recombinant protein, the recombinase of being combined with post material with 100mM imidazoles wash-out, and elution volume is 10ml.The purity of application SDS-PAGE electrophoresis detection target protein.Elution fraction is through twice 50mM, and imidazoles is removed in the 2-cyclohexylamino ethyl sulfonic acid damping fluid dialysis of pH9.0.Enzyme liquid after purifying is for kinetics and than vigor analysis.Protein concentration is measured by Bradford method, using bovine serum albumin as standard substance.
Embodiment 5: the vitality test of phosphoric triesterase
Prepare respectively various organic phosphorous insecticide solution (purchased from sigma company), the sterilant of testing is all dissolved in acetonitrile, is configured to 100mM storage liquid.The characteristic light of various organic phosphorous insecticide hydrolysates absorbs in table 2.
The characteristic light of the different organic phosphorous insecticide hydrolysates of table 2 absorbs and optical extinction coefficient
Phosphotriester enzyme activity determination is with reference to the people's such as Dumas method (Dumas, D.P., Caldwell, S.R., Wild. J.R., and Raushel, F.M. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J. Biol. Chem. 1989,264 (33): 19659-19665.).Reaction system is 1ml, buffer system is the 2-cyclohexylamino ethyl sulfonic acid damping fluid (pH9.0) of 50mM, organophosphorus substrate final concentration is 1 mM, add appropriate enzyme, 37 DEG C of reaction 10min, with UV-2550 spectrophotometer (Shimadzu company) mensuration absorbance value, not add the system of enzyme as blank.1 enzyme activity unit (U) is: at 37 DEG C, and the needed enzyme amount of per minute catalytic hydrolysis 1 μ mol substrate.The specific activity calculation formula of enzyme is as follows:
Wherein △ OD is the changing value of photoabsorption in the reaction times; V is reaction system volume (1ml); ε is optical extinction coefficient (μ M
-1); T represents the reaction times (min); V
enzymeit is the enzyme amount (ml) adding; C
enzymerepresent the concentration (mg/ml) of enzyme.
The present invention has further measured wild-type and the mutant enzyme kinetic constant for paraoxon, selected concentration of substrate scope is between 0.2-2mM, at the 2-of 50mM cyclohexylamino ethyl sulfonic acid damping fluid (pH9.0), measure enzymic activity at 37 DEG C, calculate the speed of response of enzyme.Two (Lineweaver-Burk) graphing methods reciprocal of application are to 1/V-1/[S] mapping, try to achieve K by the intercept on diaxon
mvalue (the negative inverse of x y-intercept) and V
max(inverse of y y-intercept), then by V
max=k
cat× [E] calculates k
catthereby obtain kinetic constant K
mand k
catvalue.The results are shown in Table 3, compare wild-type, the K of mutant
mvalue reduces approximately 2 times, illustrates the affinity of substrate paraoxon is improved; Gain factor k
catvalue improves approximately 133 times.Finally make catalytic efficiency (k
cat/ K
m) improve approximately 232 times.
The kinetic constant of table 3 wild-type GkaP and mutant enzyme hydrolysis paraoxon
The present invention further measured wild-type and mutant for different organic phosphorous insecticides than vigor, result is as shown in table 4.From table 4, mutant improves respectively 221,313,497 and 68 times (coordinating referring to Fig. 2) for tested paraoxon, thiophos, diazinon and chlorpyrifos than vigor.Illustrate that mutant has expanded substrate selective.
Table 4 wild-type GkaP and mutant enzyme compare vigor to several frequently seen organic phosphorous insecticide
Embodiment 6: the optimum temperuture of phosphoric triesterase wild-type and mutant thereof and pH value
The present invention has carried out the mensuration of optimal reactive temperature and pH to wild-type and mutant.Optimum temperuture be paraoxon using final concentration as 0.5mM as reaction substrate, in the temperature range of 30-100 DEG C, measure phosphotriester enzyme activity.Result shows that the optimal reactive temperature of mutant 8M of the present invention is 65 DEG C (coordinating referring to Fig. 3), compares wild-type and has declined 20 DEG C.
The mensuration of optimal pH is taking wide region pH damping fluid (composition encircle amido propanesulfonic acid, 2-code quinoline ethyl sulfonic acid for acetic acid, N-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid, N-trishydroxymethyl methyl-3-aminopropanesulfonicacid acid, 3-) as reaction system, is (6-10) damping fluid of the different pH values of 40 mM at 37 DEG C of accurate compound concentrations.Paraoxon taking final concentration as 0.5mM is reaction substrate, measures the variation of enzyme vigor under above-mentioned pH condition.Result shows that the optimum pH of wild-type enzyme is about 9; The optimum pH of mutant 8M is about 8.5-9 (coordinating referring to Fig. 4), comparing wild-type enzyme changes little, and this mutant kept more than 80% vigor in the scope of pH8-10, this wide in range pH action condition is more favourable for practical application.
Attached: the original amino acid of the phosphoric triesterase of G. kaustophilus HTA426 involved in the present invention is as shown in SEQ ID NO:1; The aminoacid sequence of mutant is as shown in SEQ ID NO:2, and the amino acid after 8 sudden changes marks by black box; The nucleotide sequence of coding G. kaustophilus HTA426 phosphoric triesterase is as shown in SEQ ID NO:3; Encode described mutant nucleotide sequence as shown in SEQ ID NO:4, and underscore represents the Nucleotide after sudden change.
<210>?SEQ?ID?NO:?1
<211>?326
<212>?PRT
The phosphotriester enzyme amino acid sequence of <213> Geobacillus kaustophilus HTA426
<400>1
<210>?SEQ?ID?NO:?2
<211>?326
<212>?PRT
The aminoacid sequence of the phosphotriester enzyme mutant of <213> Geobacillus kaustophilus HTA426
<400>2
<210>?SEQ?ID?NO:?3
<211>?981
<212>?DNA
The nucleotide sequence of the phosphoric triesterase of <213> Geobacillus kaustophilus HTA426
<400>3
<210>?SEQ?ID?NO:?4
<211>?981
<212>?DNA
The nucleotide sequence of the phosphotriester enzyme mutant of <213> Geobacillus kaustophilus HTA426
<400>4
Claims (4)
1. a phosphotriester enzyme mutant, is characterized in that, the aminoacid sequence of described mutant is as shown in SEQ ID NO.2.
2. the gene of phosphotriester enzyme mutant described in coding claim 1, is characterized in that: the nucleotide sequence of described phosphoric triesterase mutant gene is as shown in SEQ ID NO.4.
3. the application of phosphotriester enzyme mutant in degrading organic phosphor poisonous substance as claimed in claim 1.
4. the colibacillus engineering of phosphotriester enzyme mutant described in expression claim 1, is characterized in that: described colibacillus engineering (Escherichia coli) called after pET28a-GkaP (8M), deposit number is CGMCC No.5597.
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