CN102796714A - 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 phosphoric triesterase two mutants.
Background technology
Phosphoric triesterase (Phosphotriesterase; EC 3.1.8.1) organo phosphorous compounds that can the hydrolysis wide spectrum; The latter is widely used in agricultural insecticide and chemical warfare nerve poison, the phosphide key of phosphotriester endonuclease capable catalysis fracture organo phosphorous compounds and make its detoxification.Be shown below:
Organo phosphorous compounds has extremely strong toxic side effect to people and domestic animal.Organo phosphorous compounds and toxicity thereof that some are more common are seen table 1.So far have to surpass 100 kinds of organic phosphorous insecticides all over the world, account for 38% of sterilant kind being widely used.Only be used for agriculture prodn, remove and approximately produce 400,000 liters of Negasunt (coumaphos) waste liquid through " the ox flea is eradicated plan " this every year at the annual nearly 50,000 tons of organic phosphorous insecticides of the U.S.; And some European Union member countries' millions of diazinon (diazinon) that rise of production are used to clean wool liquid medicine.Because excessive and successive uses; Many soils and water resources have been suffered serious pollution; Human health has been constituted grave danger (Singh; B.K.Organophosphorus-degrading bacteria:ecology and industrial applications.Nat.Rev.Microbiol.2009,7 (2): 156-164.).Three million peoples are probably arranged in the world every year poison wherein about 200,000 people dead (Karalliedde, L. because organophosphorus is taken in; Senanayake; N., Organophosphorus insecticide poisoning.J.Int.Fed.Clin.Chem.1999,11 (2): 4-9.).On the other hand, the chemical warfare nerve poison that is used for military reserve in the world is about 200,000 tons, all destroys according to international these chemical weapons of chemical weapons treaty needs before 2023.Therefore phosphoric triesterase has wide application potential in fields such as the degraded of organophosphorus pollutent, scrubbing, biosensor, medicine research and development.
Organo phosphorous compounds that table 1 is several frequently seen and toxicity thereof
Orthogenesis is simulation Darwin nature evolutionary process in the laboratory, and to a certain proteinic encoding sox, the gene through improved induced-mutation technique transformation enzyme makes up the sudden change library, then according to specific transformation purpose, screens valuable non-natural enzyme.The orthogenesis technology obtains great success in enzymatic property transformation field, mainly concentrates on the catalytic reaction activity that improves enzyme, improves the substrate specificity of enzyme; Improve thermostability; Change the enantio-selectivity of enzyme and improve aspects such as tolerance (Turner, N.J.Directed evolution of enzymes for applied biocatalysis.Trends Biotechnol.2003,21 (11): 474-478. Johannes organic solvent; 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 people such as Takami separate in Mariana Islands submarine mud appearance and obtain; Belong to gram positive bacterium; Be kept at Japanese microbial preservation center (Japan Collection of Microorganisms; Http:// www.jcm.riken.jp/), preservation is numbered JCM 12893.It can surpass 70oC, growth (Takami, H. under the extreme condition of pH9.5-10; 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 the U.S. state-run biotechnology information center genome database.Can know that by genome sequence it contains phosphotriester enzyme coding gene (GK1506).We utilize polymerase chain reaction (Polymerization chain reaction; PCR) from G. kaustophilus HTA426 genome, increased GK1506 gene and be inserted into the pET28a expression plasmid of technology; Recombinant plasmid is transferred in the escherichia coli host; Obtain a strain engineering bacteria, called after pET28a-GkaP (WT).Engineering bacillus strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2011, be called for short CGMCC, address: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, Institute of Microorganism, Academia Sinica.Deposit number is CGMCC No.5598.Its called after of classifying: ETEC, latin name: Escherichia coli.
Technical scheme of the present invention is: through the phosphoric triesterase body variant of laboratory evolution technique construction vigor raising.The phosphoric triesterase gene (Genbank accession number 3183579) that origin comes from G. kaustophilus HTA426 carries out molecular evolution; The phosphoric triesterase expression of gene plasmid that contains to build is a template; The method of fortune fallibility PCR; Through the too much sudden change and the screening of wheel, finally obtained a phosphoric triesterase two mutants that the organic phosphorus degrading vigor significantly improves.Adopt " amino acid of original amino acid-position-sudden change " to represent the aminoacid replacement of two mutants, the amino acid mutation that contains in the said two mutants is: Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys, Gly273Asp.The colibacillus engineering strain (called after pET28a-GkaP (8M)) of expressing this two mutants has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2011; Be called for short CGMCC; Address: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, Institute of Microorganism, Academia Sinica.Deposit number is CGMCC No.5597.Its called after of classifying: ETEC, latin name: Escherichia coli.
The original amino acid of the phosphoric triesterase of G.kaustophilus HTA426 is shown in SEQ ID NO:1; The aminoacid sequence of two mutants is shown in SEQ ID NO:2, and the amino acid after 8 sudden changes marks with black box; The nucleotide sequence of coding G.kaustophilus HTA426 phosphoric triesterase wild type gene is shown in SEQ ID NO:3; Encode the nucleotide sequence of said mutant gene shown in SEQ ID NO:4, the Nucleotide after underscore is represented to suddenly change.
The expression vector that is used to express G.kaustophilus HTA426 phosphoric triesterase and two mutants thereof is pET28a; Being used for described expression vector microorganism transformed host cell is e. coli bl21 (DE3) Codon Plus.
The present invention is through protein engineering, successful acquisition the two mutants that significantly improves of organophosphorus hydrolysis vigor.Optimum two mutants is compared with wild-type enzyme, and it is for the catalytic efficiency ((k of sterilant paraoxon
Cat/ K
m) improved 232 times; 221,313,497 and 68 times have been improved for paraoxon, thiophos, diazinon, four kinds of common organic phosphorous insecticides of ENT-27311 respectively than vigor.
Description of drawings
Fig. 1 is the order-checking peak figure of phosphoric triesterase two mutants 8M (Phe28Ile/Tyr99Leu/Thr171Ser/Phe228Leu/Asn269Ser/Val270Gl y/Trp271Cys/Gly273Asp), and arrow institute labeling position is for introducing the Nucleotide of sudden change among the figure.
Fig. 2 is that wild-type GkaP and two mutants compare four kinds of common organic phosphorous insecticide vigor.
Fig. 3 is the reaction optimum temperuture of wild-type GkaP and two mutants.
Fig. 4 is the reaction ph optimum of wild-type GkaP and two mutants.
Embodiment
Used method among the following embodiment is ordinary method like no specified otherwise, and 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 that provides according to Japanese microbial preservation center is recovered and is cultivated, and at first prepares Rehydration fluid 7:5g/L peptone (peptone), 3g/L beef extract (beef extract); 10g/LNaCl; Use the deionized water constant volume, regulate pH to 7.0, autoclave sterilization.
The cell lyophilized powder of Geobacillus kaustophilus HTA426 adds 0.5ml rehydration fluid, is transferred to after the dissolving in the test tube that 5ml LB substratum is housed, and 60 ℃, 180rpm shaking culture 24 hours.Being transferred in the triangular flask that 100ml LB substratum is housed 60 ℃ then cultivated 48 hours.
Get the bacterial cultures of 5ml and collect thalline, with the resuspended deposition of 25mmol/L Tris-HCl damping fluid (pH8.0 contains 50mmol/L glucose, 10mmol/L EDTA) of 200 μ L, add the 50mg/ml N,O-Diacetylmuramidase of 50 μ L, 4 ℃ digested 1 hour; The SDS solution (final concentration is 2%W/V) that adds 125 μ L reacted 10 minutes; Add isopyknic phenol: chloroform: primary isoamyl alcohol, mixing centrifugal 5 minutes, is transferred to supernatant in another centrifuge tube; The absolute ethyl alcohol that in deposition, adds 2 times of volumes, the deposition thallus DNA, centrifugal 5 minutes of 12000rpm, remove supernatant after, the washing with alcohol DNA with 70%; Remove supernatant after centrifugal, dissolve again with the deionized water of sterilizing, gained genomic dna solution is put in-20 ℃ of refrigerators subsequent use.
(2) clone of phosphoric triesterase gene and construction of recombinant plasmid
Contain a phosphotriester enzyme coding gene (GK1506) in the Geobacillus kaustophilus HTA426 genome.The GK1506 gene is to increase from the genomic dna of above-mentioned bacterial strains through PCR method to obtain.Two primers are to cut the site according to the multienzyme of the sequence of GK1506 gene and expression plasmid to design, and it is synthetic to entrust Shanghai to give birth to worker bio-engineering corporation.
Upstream primer: 5 '-GCGC
GGATCCATGGCGGAGATGGTAGAAACGGTAT-3 ', the line part is BamH I site;
Downstream primer: 5 '-GATC
AAGCTTGTCAAGCCGAGAACAGCGCCGCCGGAT-3 ', the line part is the HindIII site.
Restriction enzyme site that two primers are set and BamH I and the HindIII of expression plasmid pET28a are complementary, and are suitable in intestinal bacteria, efficiently expressing.
PCR reaction system: in 50 μ l reaction systems, contain 1 μ l archaeal dna polymerase (the ultra fidelity dna polysaccharase of the Phusion of NEB company); 5 μ l, 10 * damping fluid; 2 μ l dNTP mixtures (every kind of nucleotide concentration 2.5mM), 2 μ l Geobacillus kaustophilus HTA426 genomic dnas, 1 μ l upstream primer (20pmol/ μ l); 1 μ l downstream primer (20pmol/ μ l), 38 μ l ultrapure waters.The PCR reaction conditions is: 95 ℃ of preparatory sex change 5min at first; Each circulation comprises 95 ℃ of sex change 40s, 58 ℃ of annealing 40s, and 72 ℃ are extended 90s, totally 30 circulations; Last 72 ℃ are extended in.Reaction finishes the back and detects the PCR product with 1% agarose gel electrophoresis.Molecular weight is consistent with (981bp) of expection.Use purification kit that the PCR product is carried out purifying.
The PCR product of purifying is cut (NEB company) with restriction enzyme BamH I and HindIII enzyme, under 37 ℃, hatched 3 hours.Enzyme cuts complete, and electrophoresis on 1.0% sepharose, Application of DNA gel reagents box reclaim the dna fragmentation after enzyme is cut.
Use same enzymic digestion pET28a plasmid, and then handle plasmid (per 50 μ l systems add 1 μ l, 37 ℃ of insulation 1h), in 0.8% sepharose, detect linear carrier and reclaim purifying with SEAP (Fermentas company).The target gene fragment that to handle then utilizes T4DNA ligase enzyme (NEB company) to be connected with the linear carrier fragment.To connect product and change among e. coli bl21 (DE3) the Codon Plus, carry out the positive colony screening with the LB solid medium that contains 50 μ g/ml kantlex.The picking mono-clonal checks order at random, and the result is consistent with report, proves the recombinant plasmid that has obtained containing the phosphoric triesterase gene, called after pET28a-GkaP.
Embodiment 2: utilize the method for fallibility PCR to make up the random mutation storehouse
Utilize the method for fallibility PCR in G.kaustophilus HTA426 phosphoric triesterase gene, to introduce the random nucleotide sudden change.Clone's phosphoric triesterase gene the primer is identical among the primer and the embodiment 1.Error-prone PCR systems is following: in 100 μ l reaction systems, contain 2 μ l Taq polysaccharases (TaKaRa company), 10 μ l, 10 * damping fluid (does not contain Mg
2+), 8 μ l dCTP (10mM), 8 μ l dTTP (10mM), 8 μ l dNTP mixtures (every kind of nucleotide concentration 2.5mM), 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 the embodiment 1.Reaction finishes the back and detects fallibility PCR product with 1% agarose gel electrophoresis.
After the fallibility pcr amplification product was purified, restriction enzyme BamH I and HindIII digested fallibility PCR product and expression plasmid pET28a respectively, connected.Connection product electric shocking method behind the purifying is transformed in e. coli bl21 (DE3) the Codon Plus competent cell.Coat on the LB resistant panel that contains the 50ug/ml kantlex, cultivated 12 hours down at 37 ℃, the whole transformants of gained are the random mutation storehouse.10 transformants of picking check order at random, the existence and the mutation rate of checking coding mutation.Transformant confirms that through order-checking the mutation rate that fallibility PCR introduces is 1-5 base/gene.
Embodiment 3: mutant choice
From sterilization toothpick picking clone the sudden change storehouse, be inoculated into every hole respectively and contain in the 96 porocyte culture plates that 200 μ l contain 50ug/ml kantlex LB liquid nutrient medium, every hole is corresponding to each specific transformant.Simultaneously, inoculation wild-type clone and empty bacterium in 96 porocyte culture plates are respectively as positive control and negative control.37 ℃, 160rpm, shaking table is cultivated about 16h, 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 ℃.
From the bacterium hole of former bacterium plate, taking out 5 μ l bacterium liquid goes to and adds 150 μ l LB liquid nutrient mediums in advance and (include the 50ug/ml kantlex, 1mM Co
2+) the corresponding aperture of 96 well culture plates in.Copy board is in 37 ℃, and the careful jolting of 180rpm is cultured to OD
600For about 0.2-0.30.Every hole adds isopropylthio β-D-galactoside (IPTG) to final concentration expresses for the 1mM inducible protein, and then in 37 ℃, the 150rpm jolting is cultured to OD
600For about 0.9-1.0, measure OD
600Ductor is put into refrigerated centrifuge, in 3000rpm, 4 ℃, centrifugal 30min, supernatant discarded.Thalline adds 50mM then in-40 ℃ of multigelations three times in every hole, the 2-cyclohexylamino ethyl sulfonic acid damping fluid of pH 9.0 (contains 0.2mM Co
2+) 200 μ l, suspension cell.In 3000rpm, 4 ℃, centrifugal 40min shifts in 100 μ l supernatant to the 96 hole enzyme plate corresponding apertures, is crude enzyme liquid.
In the 96 hole enzyme plates that contain 100 μ l crude enzyme liquids, add 100 μ l 50mM, 2-cyclohexylamino ethyl sulfonic acid damping fluid (the substrate paraoxon that the contains 0.5mM) mixing of pH 9.0,37 ℃ of reaction 10min.Reaction is measured OD after finishing immediately on ELIASA
405And preservation data.Calculate each clone OD
405/ OD
600Numerical value, contrast with the ratio of positive control, the clone who the result is higher than positive control chooses.Through continuous 4 sudden change and the screenings taken turns, the clone's quantity that amounts to screening is about 10,000.Finally obtained optimum two mutants.What relate in the aminoacid sequence of this two mutants sports Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys, Gly273Asp, and its nucleotide sequence coding order-checking peak figure sees Fig. 1.
Embodiment 4: the expression of phosphoric triesterase wild-type and two mutants thereof and purifying
The intestinal bacteria that will contain the pET28a recombinant plasmid of phosphoric triesterase (wild-type or two mutants) gene are inoculated into 5ml and contain in the LB substratum of 50ug/ml kantlex 37 ℃ of shaking culture and spend the night; Be inoculated into next day in the fresh culture of the identical resistance of 100mL, 37 ℃ are continued to cultivate 4h.The seed liquor of tentatively amplifying is inserted with 1% ratio in the LB substratum of 2L and (contain 1mM Co
2+), cultivate in the concussion of air shaking table (37 ℃, 180rpm).Treat that thalli growth is to OD
600It is 0.6～0.8 o'clock; Isopropylthio β-D-galactoside (IPTG) to the final concentration that adds 100mM is 1mM, reduces culture temperature (22 ℃), thalline is induced make 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 contains 0.2mM Co to the 50mM Tris-HCl damping fluid of 5-10 times of volume of adding
2+) resuspended thalline, ultrasonication 10 minutes; In 60 ℃ of heat treated 30 minutes, (12000rpm 30min), collected supernatant and obtains crude enzyme liquid the centrifugal intestinal bacteria foreign protein of removing sex change with broken liquid.
Because it is histidine-tagged that expressed target protein N-end contains, the application of nickel affinity column carries out purifying to recombinant protein, and with 100mM imidazoles wash-out and post material bonded recombinase, elution volume is 10ml.The purity of Using SDS-PAGE electrophoresis detection target protein.Elution fraction is through twice 50mM, and imidazoles is removed in the dialysis of the 2-cyclohexylamino ethyl sulfonic acid damping fluid of pH9.0.Enzyme liquid behind the purifying is used for kinetics and than vigor analysis.Protein concentration is measured through the Bradford method, with bovine serum albumin as standard substance.
Embodiment 5: the vitality test of phosphoric triesterase
Prepare various organic phosphorous insecticide solution (available from sigma company) respectively, the sterilant of being tested all is dissolved in the acetonitrile, is configured to the 100mM storage liquid.The characteristic light of various organic phosphorous insecticide hydrolysates absorbs sees table 2.
The characteristic light of the different organic phosphorous insecticide hydrolysates of table 2 absorbs and optical extinction coefficient
The phosphotriester enzyme activity determination is with reference to 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 1mM, adds an amount of enzyme, 37 ℃ of reaction 10min; Measure absorbance value with UV-2550 spectrophotometer (Tianjin, island company), with the system that do not add enzyme as blank.1 enzyme activity unit (U) is: at 37 ℃, and the needed enzyme amount of PM catalytic hydrolysis 1 μ mol substrate.It is following that formula is calculated in the ratio work of enzyme:
Wherein Δ 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) that adds; 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 the 0.2-2mM; At the 2-of 50mM cyclohexylamino ethyl sulfonic acid damping fluid (pH9.0), measure enzymic activity down for 37 ℃, calculate the speed of response of enzyme.Use two (Lineweaver-Burk) graphing methods reciprocal 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) is again by V
Max=k
Cat* [E] calculates k
CatThereby obtain kinetic constant K
mAnd k
CatValue.The result sees table 3, compares wild-type, the K of two mutants
mValue reduces about 2 times, explains that the affinity to the substrate paraoxon improves; Gain factor k
CatValue improves about 133 times.Finally make catalytic efficiency ((k
Cat/ K
m) improve about 232 times.
The kinetic constant of table 3 wild-type GkaP and mutant enzyme hydrolysis paraoxon
The present invention further measured wild-type and two mutants for different organic phosphorous insecticides than vigor, the result is as shown in table 4.Visible by table 4, two mutants improves 221,313,497 and 68 times (cooperating referring to Fig. 2) for the paraoxon of being tested, thiophos, diazinon and ENT-27311 respectively than vigor.Explain that two mutants has enlarged 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 two mutants thereof and pH value
The present invention has carried out the mensuration of optimal reactive temperature and pH to wild-type and two mutants.Optimum temperuture be with the final concentration be the paraoxon of 0.5mM as reaction substrate, in 30-100 ℃ TR, measure the phosphotriester enzyme activity.The result shows that the optimal reactive temperature of two mutants 8M of the present invention is 65 ℃ (cooperating referring to Fig. 3), compares wild-type and has descended 20 ℃.
The mensuration of ph optimum is to be reaction system with wide region pH damping fluid (composition is acetate, N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid, N-trishydroxymethyl methyl-3-aminopropanesulfonicacid acid, 3-cyclohexylamino propanesulfonic acid, 2-sign indicating number quinoline ethyl sulfonic acid), is (6-10) damping fluid of the different pH values of 40mM at 37 ℃ of accurate compound concentrations.The paraoxon that with the final concentration is 0.5mM is a reaction substrate, measures the variation of enzyme vigor under above-mentioned pH condition.The result shows that the optimum pH of wild-type enzyme is about 9; The optimum pH of two mutants 8M is about 8.5-9 (cooperate referring to Fig. 4), compares wild-type enzyme and changes not quite, and this two mutants kept the vigor more than 80% in the scope of pH8-10, and this wide in range pH action condition is more favourable for practical application.
Attach: the original amino acid of the phosphoric triesterase of G.kaustophilus HTA426 involved in the present invention is shown in SEQ ID NO:1; The aminoacid sequence of two mutants is shown in SEQ ID NO:2, and the amino acid after 8 sudden changes marks with black box; The nucleotide sequence of coding G.kaustophilus HTA426 phosphoric triesterase is shown in SEQ ID NO:3; Encode said two mutants nucleotide sequence shown in SEQ ID NO:4, the Nucleotide after underscore is represented to suddenly change.
<210>SEQ?IDNO：1
<211>326
<212>PRT
< 213>the phosphotriester enzyme amino acid sequence of Geobacillus kaustophilus HTA426
<400>1
Met?Ala?Glu?Met?Val?Glu?Thr?Val?Cys?Gly?Pro?Val?Pro?Val?Glu?Gln?Leu?Gly?Lys?Thr
5 10 15 20
Leu?Ile?His?Glu?His?Phe?Leu?Phe?Gly?Tyr?Pro?Gly?Phe?Gln?Gly?Asp?Val?Thr?Arg?Gly
25 30 35 40
Thr?Phe?Arg?Glu?Asp?Glu?Ser?Leu?Arg?Val?Ala?Val?Glu?Ala?Ala?Glu?Lys?Met?Lys?Arg
45 50 55 60
His?Gly?Ile?Gln?Thr?Val?Val?Asp?Pro?Thr?Pro?Asn?Asp?Cys?Gly?Arg?Asn?Pro?Ala?Phe
65 70 75 80
Leu?Arg?Arg?Val?Ala?Glu?Glu?Thr?Gly?Leu?Asn?Ile?Ile?Cys?Ala?Thr?Gly?Tyr?Tyr?Tyr
85 90 95 100
Glu?Gly?Glu?Gly?Ala?Pro?Pro?Tyr?Phe?Gln?Phe?Arg?Arg?Leu?Leu?Gly?Thr?Ala?Glu?Asp
105 110 115 120
Asp?Ile?Tyr?Asp?Met?Phe?Met?Ala?Glu?Leu?Thr?Glu?Gly?Ile?Ala?Asp?Thr?Gly?Ile?Lys
125 130 135 140
Ala?Gly?Val?Ile?Lys?Leu?Ala?Ser?Ser?Lys?Gly?Arg?Ile?Thr?Glu?Tyr?Glu?Lys?Met?Phe
145 150 155 160
Phe?Arg?Ala?Ala?Ala?Arg?Ala?Gln?Lys?Glu?Thr?Gly?Ala?Val?Ile?Ile?Thr?His?Thr?Gln
165 170 175 180
Glu?Gly?Thr?Met?Gly?Pro?Glu?Gln?Ala?Ala?Tyr?Leu?Leu?Glu?His?Gly?Ala?Asp?Pro?Lys
185 190 195 200
Lys?Ile?Val?Ile?Gly?His?Met?Cys?Gly?Asn?Thr?Asp?Pro?Asp?Tyr?His?Arg?Lys?Thr?Leu
205 210 215 220
Ala?Tyr?Gly?Val?Tyr?Ile?Ala?Phe?Asp?Arg?Phe?Gly?Ile?Gln?Gly?Met?Val?Gly?Ala?Pro
225 230 235 240
Thr?Asp?Glu?Glu?Arg?Val?Arg?Thr?Leu?Leu?Ala?Leu?Leu?Arg?Asp?Gly?Tyr?Glu?Lys?Gln
245 250 255 260
Ile?Met?Leu?Ser?His?Asp?Thr?Val?Asn?Val?Trp?Leu?Gly?Arg?Pro?Phe?Thr?Leu?Pro?Glu
265 270 275 280
Pro?Phe?Ala?Glu?Met?Met?Lys?Asn?Trp?His?Val?Glu?His?Leu?Phe?Val?Asn?Ile?Ile?Pro
285 290 295 300
Ala?Leu?Lys?Asn?Glu?Gly?Ile?Arg?Asp?Glu?Val?Leu?Glu?Gln?Met?Phe?Ile?Gly?Asn?Pro
305 310 315 320
Ala?Ala?Leu?Phe?Ser?Ala
325
<210>SEQ?ID?NO：2
<211>326
<212>PRT
< 213>aminoacid sequence of the phosphoric triesterase two mutants of Geobacillus kaustophilus HTA426
<400>2
Met?Ala?Glu?Met?Val?Glu?Thr?Val?Cys?Gly?Pro?Val?Pro?Val?Glu?Gln?Leu?Gly?Lys?Thr
5 10 15 20
Leu?Ile?His?Glu?His?Phe?Leu?
Gly?Tyr?Pro?Gly?Phe?Gln?Gly?Asp?Val?Thr?Arg?Gly
25 30 35 40
Thr?Phe?Arg?Glu?Asp?Glu?Ser?Leu?Arg?Val?Ala?Val?Glu?Ala?Ala?Glu?Lys?Met?Lys?Arg
45 50 55 60
His?Gly?Ile?Gln?Thr?Val?Val?Asp?Pro?Thr?Pro?Asn?Asp?Cys?Gly?Arg?Asn?Pro?Ala?Phe
65 70 75 80
Leu?Arg?Arg?Val?Ala?Glu?Glu?Thr?Gly?Leu?Asn?Ile?Ile?Cys?Ala?Thr?Gly?Tyr?
Tyr
85 90 95 100
Glu?Gly?Glu?Gly?Ala?Pro?Pro?Tyr?Phe?Gln?Phe?Arg?Arg?Leu?Leu?Gly?Thr?Ala?Glu?Asp
105 110 115 120
Asp?Ile?Tyr?Asp?Met?Phe?Met?Ala?Glu?Leu?Thr?Glu?Gly?Ile?Ala?Asp?Thr?Gly?Ile?Lys
125 130 135 140
Ala?Gly?Val?Ile?Lys?Leu?Ala?Ser?Ser?Lys?Gly?Arg?Ile?Thr?Glu?Tyr?Glu?Lys?Met?Phe
145 150 155 160
Phe?Arg?Ala?Ala?Ala?Arg?Ala?Gln?Lys?Glu?
Gly?Ala?Val Ile?Ile?Thr?His?Thr?Gln
165 170 175 180
Glu?Gly?Thr?Met?Gly?Pro?Glu?Gln?Ala?Ala?Tyr?Leu?Leu?Glu?His?Gly?Ala?Asp?Pro?Lys
185 190 195 200
Lys?Ile?Val?Ile?Gly?His?Met?Cys?Gly?Asn?Thr?Asp?Pro?Asp?Tyr?His?Arg?Lys?Thr?Leu
205 210 215 220
Ala?Tyr?Gly?Val?Tyr?Ile?Ala?
Asp?Arg?Phe?Gly?Ile?Gln?Gly Met?Val?Gly?Ala?Pro
225 230 235 240
Thr?Asp?Glu?Glu?Arg?Val?Arg?Thr?Leu?Leu?Ala?Leu?Leu?Arg?Asp?Gly?Tyr?Glu?Lys?Gln
245 250 255 260
Ile?Met?Leu?Ser?His?Asp?Thr?Val?
Leu?
Arg?Pro Phe?Thr?Leu?Pro?Glu
265 270 275 280
Pro?Phe?Ala?Glu?Met?Met?Lys?Asn?Trp?His?Val?Glu?His?Leu?Phe?Val?Asn?Ile?Ile?Pro
285 290 295 300
Ala?Leu?Lys?Asn?Glu?Gly?Ile?Arg?Asp?Glu?Val?Leu?Glu?Gln?Met?Phe?Ile?Gly?Asn?Pro
305 310 315 320
Ala?Ala?Leu?Phe?Ser?Ala
325
<210>SEQ?ID?NO：3
<211>981
<212>DNA
< 213>nucleotide sequence of the phosphoric triesterase of Geobacillus kaustophilus HTA426
<400>3
1 ATGGCGGAGA?TGGTAGAAAC?GGTATGCGGG?CCGGTGCCGG?TGGAACAGCT
51?TGGCAAAACG?CTCATCCACG?AGCATTTCCT?CTTCGGTTAT?CCAGGGTTTC
101AAGGCGATGT?GACGCGCGGC?ACGTTCCGTG?AAGACGAGTC?GCTTCGCGTC
151GCAGTCGAGG?CGGCGGAAAA?GATGAAGCGG?CACGGCATTC?AAACGGTTGT
201CGATCCGACG?CCGAACGATT?GCGGGCGCAA?CCCGGCGTTT?TTGCGGCGCG
251TCGCTGAAGA?GACGGGGCTG?AACATTATTT?GCGCCACCGG?CTATTATTAT
301GAAGGGGAAG?GGGCGCCGCC?GTACTTCCAA?TTCCGCCGGC?TTCTCGGAAC
351AGCAGAAGAT?GACATTTACG?ACATGTTTAT?GGCCGAGCTG?ACCGAGGGCA
401TTGCCGATAC?CGGAATCAAG?GCGGGTGTCA?TCAAGCTCGC?CTCGAGCAAA
451GGGCGCATCA?CCGAGTACGA?AAAGATGTTC?TTCCGCGCCG?CCGCCCGCGC
501GCAAAAAGAG?ACGGGCGCGG?TCATCATCAC?CCATACGCAA?GAAGGAACGA
551TGGGGCCGGA?ACAAGCCGCC?TATTTGCTTG?AGCACGGCGC?CGATCCGAAA
601AAAATTGTCA?TCGGCCATAT?GTGCGGCAAC?ACGGACCCGG?ACTATCATCG
651AAAGACGCTT?GCTTACGGCG?TTTACATTGC?GTTTGACCGC?TTCGGCATCC
701AAGGGATGGT?CGGCGCGCCG?ACCGATGAGG?AGCGGGTGCG?GACGCTCCTT
751GCTCTGCTCC?GCGATGGGTA?CGAGAAACAA?ATTATGCTGT?CGCATGACAC
801TGTCAACGTT?TGGCTCGGTC?GTCCGTTTAC?GCTGCCGGAA?CCGTTTGCGG
851AAATGATGAA?AAATTGGCAT?GTCGAGCATT?TGTTTGTGAA?CATCATCCCC
901GCGCTGAAAA?ATGAAGGAAT?CCGCGACGAA?GTGCTTGAGC?AAATGTTCAT
951CGGCAATCCG?GCGGCGCTGT?TCTCGGCTTG?A
<210>SEQ?ID?NO：4
<211>981
<212>DNA
< 213>nucleotide sequence of the phosphoric triesterase two mutants of Geobacillus kaustophilus HTA426
<400>4
1 ATGGCGGAGA?TGGTAGAAAC?GGTATGCGGG?CCGGTGCCGG?TGGAACAGCT
51?TGGCAAAACG?CTCATCCACG?AGCATTTCCT?C
ATCGGTTAT?CCAGGGTTTC
101AAGGCGATGT?GACGCGCGGC?ACGTTCCGTG?AAGACGAGTC?GCTTCGCGTC
151GCAGTCGAGG?CGGCGGAAAA?GATGAAGCGG?CACGGCATTC?AAACGGTTGT
201CGATCCGACG?CCGAACGATT?GCGGGCGCAA?CCCGGCGTTT?TTGCGGCGCG
251TCGCTGAAGA?GACGGGGCTG?AACATTATTT?GCGCCACCGG?CTAT
CTTTAT
301GAAGGGGAAG?GGGCGCCGCC?GTACTTCCAA?TTCCGCCGGC?TTCTCGGAAC
351AGCAGAAGAT?GACATTTACG?ACATGTTTAT?GGCCGAGCTG?ACCGAGGGCA
401TTGCCGATAC?CGGAATCAAG?GCGGGTGTCA?TCAAGCTCGC?CTCGAGCAAA
451GGGCGCATCA?CCGAGTACGA?AAAGATGTTC?TTCCGCGCCG?CCGCCCGCGC
501GCAAAAAGAG?
TCGGGCGCGG?TCATCATCAC?CCATACGCAA?GAAGGAACGA
551TGGGGCCGGA?ACAAGCCGCC?TATTTGCTTG?AGCACGGCGC?CGATCCGAAA
601AAAATTGTCA?TCGGCCATAT?GTGCGGCAAC?ACGGACCCGG?ACTATCATCG
651AAAGACGCTT?GCTTACGGCG?TTTACATTGC?GTT
AGACCGC?TTCGGCATCC
701AAGGGATGGT?CGGCGCGCCG?ACCGATGAGG?AGCGGGTGCG?GACGCTCCTT
751GCTCTGCTCC?GCGATGGGTA?CGAGAAACAA?ATTATGCTGT?CGCATGACAC
801TGTCA
GCG
GT?TG
CCTCG
ATC?GTCCGTTTAC?GCTGCCGGAA?CCGTTTGCGG
851AAATGATGAA?AAATTGGCAT?GTCGAGCATT?TGTTTGTGAA?CATCATCCCC
901GCGCTGAAAA?ATGAAGGAAT?CCGCGACGAA?GTGCTTGAGC?AAATGTTCAT
951CGGCAATCCG?GCGGCGCTGT?TCTCGGCTTG?A
Claims (6)
1. phosphoric triesterase two mutants; It is characterized in that; The aminoacid sequence of said two mutants wherein comprises following 8 amino acid mutation: Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys, Gly273Asp shown in SEQ ID NO.2.
2. the preparation method of phosphoric triesterase two mutants as claimed in claim 1; It is characterized in that: the phosphoric triesterase gene that origin comes from G.kaustophilus HTA426 carries out molecular evolution; The phosphoric triesterase expression of gene plasmid that contains to build is a template; The method of utilization fallibility PCR through the too much sudden change and the screening of wheel, finally obtains the phosphoric triesterase two mutants; The aminoacid sequence of said phosphoric triesterase wild-type is shown in SEQ ID NO.1.
3. coding claim 1 said phosphoric triesterase wild type gene, it is characterized in that: the nucleotide sequence of said phosphoric triesterase wild type gene is shown in SEQ ID NO.3.
4. coding claim 1 said phosphoric triesterase mutant gene, it is characterized in that: the nucleotide sequence of said phosphoric triesterase mutant gene is shown in SEQ ID NO.4.
5. the application of phosphoric triesterase two mutants in the degrading organic phosphor poisonous substance according to claim 1.
6. express the colibacillus engineering of the said phosphoric triesterase two mutants of claim 1, it is characterized in that: said colibacillus engineering called after pET28a-GkaP (8M), deposit number are CGMCC No.5597.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109402222A (en) * | 2018-02-05 | 2019-03-01 | 中国科学院青岛生物能源与过程研究所 | The high-throughput screening method of hydrolase |
| CN111394335A (en) * | 2020-04-13 | 2020-07-10 | 中国农业科学院研究生院 | Phosphotriesterase Sy-PTE and application thereof in degradation of organic phosphorus flame retardant |
| CN112430585A (en) * | 2021-01-27 | 2021-03-02 | 凯莱英生命科学技术(天津)有限公司 | Esterase mutant and application thereof |
| CN113677207A (en) * | 2019-03-19 | 2021-11-19 | 地中海感染基金会 | Novel use and composition of mutant lactonase |
| CN114302959A (en) * | 2019-06-17 | 2022-04-08 | 米加尔-加利里研究院有限公司 | Stable mutants of quorum sensing quenched lactonases and their use in the treatment of pathogens |
| CN114302954A (en) * | 2019-04-08 | 2022-04-08 | 新加坡国立大学 | Use of synthetic lixiviant biology for the recovery of precious and toxic metals from man-made sources |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402222A (en) * | 2018-02-05 | 2019-03-01 | 中国科学院青岛生物能源与过程研究所 | The high-throughput screening method of hydrolase |
| CN113677207A (en) * | 2019-03-19 | 2021-11-19 | 地中海感染基金会 | Novel use and composition of mutant lactonase |
| CN113677207B (en) * | 2019-03-19 | 2023-10-13 | 地中海感染基金会 | New use and composition of mutant lactonase |
| CN114302954A (en) * | 2019-04-08 | 2022-04-08 | 新加坡国立大学 | Use of synthetic lixiviant biology for the recovery of precious and toxic metals from man-made sources |
| CN114302959A (en) * | 2019-06-17 | 2022-04-08 | 米加尔-加利里研究院有限公司 | Stable mutants of quorum sensing quenched lactonases and their use in the treatment of pathogens |
| CN111394335A (en) * | 2020-04-13 | 2020-07-10 | 中国农业科学院研究生院 | Phosphotriesterase Sy-PTE and application thereof in degradation of organic phosphorus flame retardant |
| CN112430585A (en) * | 2021-01-27 | 2021-03-02 | 凯莱英生命科学技术(天津)有限公司 | Esterase mutant and application thereof |
| WO2022160408A1 (en) * | 2021-01-27 | 2022-08-04 | 凯莱英生命科学技术(天津)有限公司 | Esterase mutant and use thereof |
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