CN102643841B - Fenoxaprop-p-ethyl hydrolysis esterase gene, engineering bacteria containing such gene and application of fenoxaprop-p-ethyl hydrolysis esterase gene - Google Patents
Fenoxaprop-p-ethyl hydrolysis esterase gene, engineering bacteria containing such gene and application of fenoxaprop-p-ethyl hydrolysis esterase gene Download PDFInfo
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- PQKBPHSEKWERTG-LLVKDONJSA-N ethyl (2r)-2-[4-[(6-chloro-1,3-benzoxazol-2-yl)oxy]phenoxy]propanoate Chemical group C1=CC(O[C@H](C)C(=O)OCC)=CC=C1OC1=NC2=CC=C(Cl)C=C2O1 PQKBPHSEKWERTG-LLVKDONJSA-N 0.000 title claims abstract description 90
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- 239000005502 Cyhalofop-butyl Substances 0.000 claims description 8
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- 239000005614 Quizalofop-P-ethyl Substances 0.000 claims description 8
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Abstract
The invention belongs to the field of genetic engineering, and relates to a fenoxaprop-p-ethyl hydrolysis esterase gene, engineering bacteria containing such gene and application of the fenoxaprop-p-ethyl hydrolysis esterase gene. The fenoxaprop-p-ethyl hydrolysis esterase gene has a nucleotide sequence of SEQ ID NO. 1. Fenoxaprop-p-ethyl hydrolysis esterase gene protein coded by the fenoxaprop-p-ethyl hydrolysis esterase gene has an amino acid sequence of SEQ ID NO. 2. The fenoxaprop-p-ethyl hydrolysis esterase gene is constructed into a recombinant plasmid and then introduced into E. coli BL21 (DE3) to obtain the genetic engineering bacteria comprising the gene. The fenoxaprop-p-ethyl hydrolysis esterase gene can be applied to the aspects such as hydrolytic aryloxyphenoxypropionate herbicides, triacylglycerol and p-nitrophenyl ester. An enzyme preparation produced by using the gene can be used in the industries such as biological remediation of environment, food processing, medicine and washing; the problem of environment pollution of the fenoxaprop-p-ethyl can be solved; and considerable economic benefit can also be achieved.
Description
Technical field
The invention belongs to the genetically engineered field, relate to fenoxapropPethyl hydrolysis esterase gene, the engineering bacteria that contains this gene and application thereof.
Background technology
FenoxapropPethyl 2-[4-(6-chloro-1,3-benzoxazoles-2-oxygen base) phenoxy group] ethyl propionate be 1978 by the existing merger of German Hoechst(in German Bayer) company, a kind of fragrant oxygen phenoxy propionic acid weedicide of preventing and kill off gramineous weeds that develops, belong to the inner sucting conduction type selective post-emergence herbicide, be mainly used in the multiple gramineous weedss such as wheatland Control Wild oat, amur foxtail, the annual and perennial gramineous weeds that also can prevent and kill off beans, beet, cotton, flax, peanut and vegetable fields.At present in the world and China use very wide.But studies show that in a large number fenoxapropPethyl shows as high toxicity to hydrobiont.
The degraded of environmental pollutant mainly depends on the effect of Soil Microorganism, and microorganism is mainly finished by intracellular enzyme the degraded of material.Microorganism is the important sources of various enzymes.By from environment, separating microbes producing cellulase, utilize molecule clone technology from microbes producing cellulase, to clone to produce the enzyme gene, again it is connected with suitable carrier and changes corresponding host cell over to, can carry out the great expression of enzyme.At present, produce enzyme by the genetic engineering technique means, become the leading of industrial enzymes.
Esterase (esterase) broadly refers to have the general name of a class lytic enzyme of catalytic hydrolysis ester bond ability, and usually said esterase Procaine esterase (carboxylesterase often, E.C.3.1.1.1), be mainly used to hydrolysed fat acid family and aromatic series Ester.Triglyceride class and α-naphthylacetate and p-nitrophenyl Ester all are the specific substrates of esterase.Because esterase has very high activity, as biological catalyst, utilize its hydrolysis reaction, ester conversion and ester synthesis reaction can be widely used in medicine, chemical industry, food, the energy and field of Environment Protection.
Summary of the invention
The objective of the invention is to produce highly toxic problem for fenoxapropPethyl to hydrobiont, a kind of fenoxapropPethyl hydrolysis esterase gene is provided.
Another object of the present invention provides the genetic engineering bacterium that contains this gene.
Another purpose of the present invention provides the application of this gene.Utilize the zymin of this genes produce to can be used for the industry such as biological restoration, food-processing, medicine, washing of environment, not only can solve the problem of environmental pollution of fenoxapropPethyl, can also obtain considerable economic benefit.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of fenoxapropPethyl hydrolysis esterase gene, its nucleotides sequence is classified SEQ ID NO.1 as.This full length gene (from the initiator codon to the terminator codon) is 1140bp, and G+C content is 63.07%, 379 amino acid of encoding, and its aminoacid sequence is: SEQ ID NO.2.
The coded fenoxapropPethyl ester hydrolysis zymoprotein of described fenoxapropPethyl hydrolysis esterase gene nucleotide series, its aminoacid sequence is: SEQ ID NO.2.
A kind of recombinant plasmid, this recombinant plasmid are pET-29a (+) recombinant plasmids that contains described fenoxapropPethyl hydrolysis esterase gene.
The genetic engineering bacterium E.coli BL21 (DE3) that contains described fenoxapropPethyl hydrolysis esterase gene.
The construction process of described genetic engineering bacterium E.coli BL21 (DE3): described pET-29a (+) recombinant plasmid transformed that contains fenoxapropPethyl hydrolysis esterase gene obtains recombinant microorganism E.coli BL21 (DE3) to expressive host bacterium E.coli BL21 (DE3), again the recombinant microorganism E.coli BL21 (DE3) that obtains is transferred to and contains the 200mg/l fenoxapropPethyl, the flat board of 50mg/l kantlex and 24mg/l IPTG, behind 37 ℃ of cultivation 16h, picking has the positive transformant of hydrolysis, after the sequence verification gene order is errorless, preserve.
The genetically engineered of described fenoxapropPethyl hydrolysis esterase gene aspect the hydrolysis of fragrant phenoxy phenoxy propionic acid weedicide used.
Wherein, the preferred fenoxapropPethyl of described fragrant phenoxy phenoxy propionic acid weedicide, quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl.
The application of described fenoxapropPethyl hydrolysis esterase gene in the hydrolysis triglyceride, the preferred vanay of described triglyceride, tributyrin or tricaproin.
The application of described fenoxapropPethyl hydrolysis esterase gene in the hydrolysis p-nitrophenyl ester, the preferred acetic acid p-nitrophenyl acetate of described p-nitrophenyl ester, butyric acid p-nitrophenyl ester, caproic acid p-nitrophenyl ester, sad p-nitrophenyl ester, capric acid p-nitrophenyl ester, lauric acid p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester, palmitinic acid p-nitrophenyl ester, further preferred butyric acid p-nitrophenyl ester.
The application of described fenoxapropPethyl ester hydrolysis zymoprotein aspect the fragrant phenoxy phenoxy propionic acid weedicide of hydrolysis.
Wherein, described fragrant phenoxy phenoxy propionic acid weedicide is preferred fenoxapropPethyl, quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl.
The application of described fenoxapropPethyl ester hydrolysis zymoprotein in the hydrolysis triglyceride, the preferred vanay of described triglyceride, tributyrin or tricaproin.
The application of described fenoxapropPethyl ester hydrolysis zymoprotein in the hydrolysis p-nitrophenyl ester, the preferred acetic acid p-nitrophenyl acetate of described p-nitrophenyl ester, butyric acid p-nitrophenyl ester, caproic acid p-nitrophenyl ester, sad p-nitrophenyl ester, capric acid p-nitrophenyl ester, lauric acid p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester, palmitinic acid p-nitrophenyl ester, further preferred butyric acid p-nitrophenyl ester.
Beneficial effect of the present invention is as follows:
The present invention take fenoxapropPethyl as sole carbon source from soil separation screening to bacterial strain rhodococcus (Rhodococcus sp.) T1 of a high-efficiency degradation fenoxapropPethyl, and take this bacterial strain as material construction its genomic library, and successfully from this library, clone fenoxapropPethyl hydrolysis esterase (one of key enzyme in the fenoxapropPethyl degradation process) gene.The product fenoxapropPethyl hydrolysis esterase of this genetic expression can be hydrolyzed fragrant phenoxy phenoxy propionic acid Herbicide Jing fenoxaprop-P, quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl etc. efficiently.
2. utilize this gene constructed engineering strain to be hydrolyzed esterase by high efficient expression fenoxapropPethyl, this esterase can also act on other ester compounds such as triglyceride, p-nitrophenyl ester, and the zymin of production can be used for the industries such as the biological restoration of environment, food-processing, medicine and washing.
Description of drawings
The policy map of Fig. 1 fenoxapropPethyl hydrolysis esterase gene clone.
Fig. 2 fenoxapropPethyl hydrolysis esterase gene is at E.coli BL21(pET-29a (+)) in high efficient expression experimental program figure.
Biomaterial preservation information
Rhodococcus (Rhodococcus sp.) T1 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on July 5th, 2011, the address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.5030.
Embodiment
Embodiment 1. fenoxapropPethyls hydrolysis esterase gene cloning
1.1 the screening of rhodococcus (Rhodococcus sp.) T1
Enrichment medium (the K that contains the 25mg/l fenoxapropPethyl at 100ml
2HPO
41.5g/l, KH
2PO
40.5g/l, NH
4NO
31.0g/l, MgSO
47H
2O 0.10g/l, NaCl 1.0g/l, yeast powder 20mg/l, pH 7.0) in add the soil sample that 5g is polluted by fenoxapropPethyl, cultivate 7d in 30 ℃, 180rpm.Enrichment culture liquid after hcl acidifying and methylene dichloride extracting, utilize high-performance liquid chromatogram determination that degradation effect is arranged after, draw the 10ml pregnant solution and be forwarded in the fresh enrichment medium, continuously enrichment, and the concentration that progressively improves fenoxapropPethyl is to 200mg/l.After the pregnant solution degradation effect is stable, adopt the gradient dilution flat band method, the pregnant solution that dilutes is coated with 30 ℃ of cultivations at the MSM solid medium flat board that contains the 100mg/l fenoxapropPethyl.Behind the single bacterium colony of dull and stereotyped upper appearance, there is single bacterium colony of transparent circle to be forwarded to the liquid minimal medium (K that contains the 25mg/l fenoxapropPethyl around the choosing colony
2HPO
41.5g/l, KH
2PO
40.5g/l, NH
4NO
31.0g/l, MgSO
47H
2O 0.10g/l, NaCl1.0g/l, pH 7.0) in, 30 ℃, 180rpm are cultivated 2d.Nutrient solution is measured single bacterium to the degradation effect of fenoxapropPethyl through hcl acidifying after the methylene dichloride extracting.Wherein bacterial strain T1 has the ability of the strongest degraded fenoxapropPethyl, is accredited as Rhodococcus sp. through physiological and biochemical test and 16S rDNA sequence amplification, order-checking.This bacterial strain in the LB of pH 7.0 substratum, 30 ℃ of lower well-growns.Cultivate the visible oyster white of 24h or incarnadine circular, moistening, the bacterium colony of thickness.Rhodococcus (Rhodococcus sp.) T1 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on July 5th, 2011, the address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.5030.
1.2 the structure of rhodococcus (Rhodococcus sp.) T1 genomic library
Adopt high salt method T1(CGMCC No.5030) the total DNA of karyomit(e), Sau3AI is partially digested with it with restriction enzyme, again through the dna fragmentation of agarose gel electrophoresis Separation and Recovery 4-6kb, with dephosphorization carrier the pUC118 ((TaKaRa of BamH I/BAP), Code:D3321, down together) after the connection, chemical conversion imports the E.coli DH5 α competent cell (TaKaRa for preparing, Code:D9057, lower same), make up rhodococcus (Rhodococcus sp.) T1 genomic library.Select the positive colony that contains fenoxapropPethyl hydrolysis esterase gene by the LB flat board that contains the 200mg/l fenoxapropPethyl.
1.2.1 the extraction of the total DNA of thalline
Adopt high salt method rhodococcus (Rhodococcus sp.) T1(CGMCC No.5030) the total DNA of karyomit(e): picking rhodococcus (Rhodococcus sp.) T1(CGMCC No.5030) single colony inoculation is in 3ml LB liquid nutrient medium, 30 ℃, 180rpm are cultured to OD600nm ≈ 1.0, the centrifugal collection thalline of 12000rpm; With 1.0mL TE damping fluid (10mmol/l TrisCl (pH8.0), 1mmol/l EDTA, pH 8.0) resuspended washing thalline, the centrifugal 5min of 10000rpm collects thalline, add 1.0ml TEN damping fluid (10mmol/l TrisCl (pH8.0), 1mmol/l EDTA, 0.1mol/l NaCl pH 8.0) suspension thalline, the N,O-Diacetylmuramidase (100mg/mL) that adds 5 μ l, 37 ℃ of water-bath 1h, add 25-50 μ l 20%SDS and 5 μ l Proteinase Ks (20mg/ml), 65 ℃ of water-bath 2h, after the liquid clarification, add the saturated NaCl solution of 340 μ l concuss, the centrifugal 10min of 12000rpm, supernatant liquor is transferred in the aseptic clean eppendorf pipe, use isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) extracting is clarified without till the white solid thing to the interface, shift supernatant liquor in another aseptic clean eppendort pipe, add the Virahol of 0.6 times of volume, place 0.5-1h precipitation DNA for-20 ℃, the centrifugal 10min of 12000rpm, remove behind the supernatant with 70% washing with alcohol 2 times, after the ethanol volatilization, add 30 μ l sterilized water or TER, place 4 ℃ of refrigerator overnight and dissolve, 4 ℃ of preservations of short-term ,-20 ℃ of long-term employings are frozen.
1.2.2 it is as follows that restriction enzyme Sau3AI enzyme is cut total DNA system:
37 ℃ of enzymes are cut 30min.Add 3 μ l, 10 * loading buffer and stop endonuclease reaction.Enzyme is cut product and is separated through 0.75% agarose gel electrophoresis, reclaims the 4-6kb dna fragmentation.Recovery is carried out according to the test kit specification sheets.
1.2.3 endonuclease bamhi and dephosphorization carrier pUC118 (being connected of BamH I/BAP)
(the BamH I/BAP) carrier DNA is transferred in the aseptic Eppendorf tube with 1 μ l pUC118, the Sau3AI enzyme that adds the total DNA of 6 μ l cuts back to close fragment, add water to 8.5 μ l, in 45 ℃ of 5min that heat the cohesive end of again annealing is unwind, mixture is cooled to 0 ℃.Then add 1 μ l10 * T
4The dna ligase damping fluid, 0.5 μ l T
4Dna ligase.More than 16 ℃ of ligation 12h.
1.3 enzyme connects the conversion of product and the screening of positive colony
10 μ l enzymes are connected product join in the E.coli DH5 α competent cell of 200 μ l after melting on ice, ice bath 30min is in 42 ℃ of water-baths behind the heat shock 90s.Fast transfer cools off 1 ~ 2min in ice bath, add 800 μ l liquid LB substratum, 37 ℃ of shaking table 80-90rpm incubation 45min, recovery cell in every pipe.The centrifugal 3min of 4000rpm, remaining 200 μ l competent cells coats on the LB agar plate that contains 200mg/l fenoxapropPethyl and 100mg/l penbritin, flat-plate inverted places 37 ℃ of incubators to cultivate, bacterium colony appears behind the 12-16h, selection has clone's of hydrolysis circle, be positive colony that contains fenoxapropPethyl hydrolysis esterase gene fragment, simultaneously positive colony be numbered.Choose single colony inoculation in 3ml LB liquid tube, upgrading grain checking after growing bacteria suspension.
1.4 the order-checking of the sub-Insert Fragment of positive colony and analysis
The order-checking of the sub-Insert Fragment of positive colony entrusts the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd to finish.Sequencing primer is pUC118 (Auele Specific Primer on the carrier of BamH I/BAP), and according to two ends sequencing result design primer, until whole Insert Fragment is surveyed logical.
The sequence that order-checking is finished is compared in ncbi database, and utilize ORF finder instrument to identify the contained ORF of Insert Fragment.
1.5 the pcr amplification of fenoxapropPethyl hydrolysis esterase gene
FenoxapropPethyl hydrolysis esterase ORF according to inferring designs two pairs of primers, utilizes overlap extension pcr amplification fenoxapropPethyl hydrolysis esterase gene.Wherein forward primer F1:5 '-
CATATGGCGAACATCGAAGGCGTA-3 ' (SEQ ID NO.3) is with the NdeI restriction enzyme site, reverse primer R1:5 '-
CTCGAGTCAACTCAAAGCGTCGTAGGC-3 ' (SEQ ID NO.4) is used for the full gene of amplification fenoxapropPethyl hydrolysis esterase with an XhoI restriction enzyme site.Other pair of primers F2:5 '-GTTCACC
CTGGAGAACATTTG-3 ' (SEQ ID NO.5) and R2:5 '-CAAATGTT
CTGGAGGGTGAAC-3 ' (SEQ ID NO.6) is a pair of complementary sequence, is used for making an XhoI restriction enzyme site generation rite-directed mutagenesis of fenoxapropPethyl hydrolysis esterase gene inside, avoids fenoxapropPethyl hydrolysis esterase gene to be cut off by XhoI.
The PCR reaction divides to be carried out for four times, for the first time in 1.4 through the positive colony plasmid that contains fenoxapropPethyl hydrolysis esterase gene of sequence verification as template, utilize primers F 1 and R2 to increase to comprising the fragment of XhoI restriction enzyme site in interior left side in the fenoxapropPethyl hydrolysis esterase gene.Utilize for the second time primers F 2 and R1 to comprise the fragment of XhoI restriction enzyme site on interior right side as the primer pair fenoxapropPethyl is hydrolyzed in the esterase gene increases take the positive colony plasmid as template.The PCR product of twice amplification is template for the third time, utilizes primers F 1 and R1 to increase, and obtains the complete genome of fenoxapropPethyl hydrolysis esterase.Utilize the Taq enzyme that amplified fragments is added the A tail take amplified production for the third time as template the 4th time.
For the first time amplification:
Amplification system:
The pcr amplification program:
A.98 ℃ sex change 10sec, 55 ℃ of annealing 10sec, 72 ℃ are extended 1min, carry out 30 circulations;
B.72 ℃ extend 10min;
C.15 ℃ cooling 10min.
For the second time amplification: change amplimer into F2 and R1, all the other increase identical with the first time.
For the third time amplification:
Amplification system:
The pcr amplification program:
The 4th amplification: the PCR product adds the A tail
Amplification system:
The pcr amplification condition: 72 ℃ are extended 20min; 10 ℃ of insulation 10min.
1.6 enzyme connects
The fenoxapropPethyl ester hydrolysis enzyme dna fragment and pMD 19-T Vector(TaKaRa Code:D102A that add the A tail through PCR) in molar ratio 3:1 mix, under the connecting fluid effect, 16 ℃ of water-baths are spent the night.The enzyme disjunctor is as follows:
1.7 enzyme connects the conversion of product and the screening of positive colony (with reference to 1.3)
1.8 the extraction of the sub-plasmid of positive colony and order-checking
Screen the positive colony overnight incubation in containing the LB substratum of penbritin that obtains in 1.7, the centrifugal 10min of 12000rpm collects thalline, utilizes plasmid extraction kit to extract plasmid, serves the order-checking of the prompt basic biological company limited in the extra large English Weihe River.
The high efficient expression of embodiment 2. fenoxapropPethyls hydrolysis esterase gene in E.coli BL21 (pET-29a (+))
2.1 with the recombinant plasmid that extracts in 1.8 with NdeI and XhoI double digestion
Enzyme is cut system:
In 37 ℃ of water-baths, more than the reaction 3h.Enzyme is cut product and is carried out 0.75% agarose gel electrophoresis and cut glue and reclaim.
(2.2pET-29a+) (Merck-Novagen, Cat NO.69871) NdeI and XhoI double digestion (with reference to 2.5).
2.3 transform and express
2.1 in recovery fragment and 2.2 in the pET-29a (+) that cuts of enzyme carry out enzyme and get pET-29a (+) recombinant plasmid that contains fenoxapropPethyl hydrolysis esterase gene continuously.
Enzyme connects good pET-29a (+) recombinant plasmid transformed that contains fenoxapropPethyl hydrolysis esterase gene to expressive host bacterium E.coli BL21 (DE3) (NBE, Cat NO.C2527H) obtains recombinant microorganism E.coli BL21 (DE3), coating contains the flat board of 200mg/l fenoxapropPethyl, 50mg/l kantlex and 24mg/l IPTG, behind 37 ℃ of cultivation 16-20h, picking has positive colony of hydrolysis, and is errorless through the sequence verification gene order.
2.4 the enzyme that the checking positive transformant is expressed is to the hydrolysis function of fenoxapropPethyl
2.3 in positive colony 37 ° of C in the LB substratum of obtaining be cultured to 0D
600nmBetween 0.5-0.6, add IPTG to concentration 0.2mM, 18 ℃ are continued to cultivate 24h.After the collection thalline was resuspended with phosphoric acid buffer (pH8.0), with the broken somatic cells of supersound process, the centrifugal 15min of 20000g, gained supernatant were fenoxapropPethyl hydrolysis esterase crude enzyme liquid.Get 10 μ l fenoxapropPethyls hydrolysis esterase crude enzyme liquid and add in the phosphoric acid buffer that 4ml contains the 100mg/l fenoxapropPethyl, behind 50 ℃ of reaction 5min, detect the degraded situation of fenoxapropPethyl with high performance liquid chromatography.The enzyme unit definition of living is that per minute produces 1 μ mol fenoxapropPethyl acid and is a unit, the ratio vigor that records thick enzyme is 31.83 μ mol/min/mg, namely this thick enzyme of 1mg take fenoxapropPethyl as substrate under optimum condition the reaction per minute can obtain 31.83 μ mol fenoxapropPethyls acid.After this enzyme is 2995.03 μ mol/min/mg by the Ni-NTA affinitive layer purification and through recording the ratio vigor of this enzyme take fenoxapropPethyl as substrate the time behind the ultrafiltration and concentration, namely the pure enzyme of 1mg per minute when fenoxapropPethyl is substrate produces 2995.03 μ mol fenoxapropPethyls acid.This result shows that the ratio enzyme activity after purifying concentrates is about 94 times of thick enzyme.
Embodiment 3 fenoxapropPethyls hydrolysis esterase is to the hydrolysis of triglyceride
The fenoxapropPethyl hydrolysis esterase (preparation method is with embodiment 2) of 337 μ g purifying is joined the phosphoric acid buffer (0.02mol/l that 5ml contains respectively 0.02mol/l vanay, tributyrin, tricaproin, caprylin and caprin, pH7.5) in, at 50 ℃ of lower reaction 10min, add again two 1% phenolphthalein solutions after adding 10ml ethanol termination reaction, be titrated to lightpink with 0.01mol/l NaOH, record NaOH consumption.The enzyme unit definition of living is that per minute produces 1 μ mol respective acids and is a unit.The result shows that fenoxapropPethyl hydrolysis esterase can act on vanay, tributyrin and tricaproin, and their ratio vigor is respectively 3164.47,1107.57 and 59.33 μ mol/min/mg.But can not act on caprylin and caprin.
Embodiment 4 fenoxapropPethyls hydrolysis esterase is to the hydrolysis of p-nitrophenyl ester
The fenoxapropPethyl hydrolysis esterase (preparation method is with embodiment 2) of 0.67 μ g purifying is joined the phosphoric acid buffer (0.02mol/l that 4ml contains respectively 0.02mol/l acetic acid p-nitrophenyl acetate, butyric acid p-nitrophenyl ester, caproic acid p-nitrophenyl ester, sad p-nitrophenyl ester, capric acid p-nitrophenyl ester, lauric acid p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester and palmitinic acid p-nitrophenyl ester, pH8.0) in, 50 ℃ of lower reactions, dilute immediately 10 times behind the 5min, measure OD
410nmThe enzyme unit definition of living is that per minute produces 1 μ mol respective acids and is a unit.The result shows that fenoxapropPethyl hydrolysis esterase is the strongest to the hydrolysis vigor of butyric acid p-nitrophenyl ester, is 13814.16 μ mol/min/mg than vigor.Prolongation along with fatty acid carbon chain, the hydrolysis vigor reduces gradually, and its ratio vigor to acetic acid p-nitrophenyl acetate, caproic acid p-nitrophenyl ester, sad p-nitrophenyl ester, capric acid p-nitrophenyl ester, lauric acid p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester and palmitinic acid p-nitrophenyl ester is respectively 7013.14,9011.83,5627.81,2542.37,1148.951,199.06 and 130.63 μ mol/min/mg.
Embodiment 5 fenoxapropPethyls hydrolysis esterase is to the hydrolysis of other fragrant phenoxy phenoxy propionic acid weedicides
The fenoxapropPethyl of 1.47 μ g purifying hydrolysis esterase (preparation method is with embodiment 2) is joined in the phosphoric acid buffer that 4ml contains respectively 100mg/l quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl (0.02mol/l, pH8.0), at 50 ℃ of lower reactions, 5min.Add 10%HCl and regulate pH to 2.0, add the extracting of equal-volume methylene dichloride.The phase of anhydrating adds anhydrous sodium sulphate and removes residual moisture.Get the 1ml extract, room temperature evaporates into dried.Residuum with dissolve with methanol after, detect the degraded situation of quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl with high performance liquid chromatography.The enzyme unit definition of living is that the per minute corresponding substrate of 1 μ mol of degrading is a unit, records pure enzyme the ratio vigor of quizalofopPethyl, clodinafop-propargyl and cyhalofop-butyl is respectively 3287,3463,2496 μ mol/min/mg.
Claims (10)
1. fenoxapropPethyl hydrolysis esterase gene is characterized in that nucleotides sequence classifies as: SEQ ID
NO.1
2. fenoxapropPethyl claimed in claim 1 is hydrolyzed the fenoxapropPethyl ester hydrolysis zymoprotein of esterase coded by said gene, and its aminoacid sequence is: SEQ ID
NO.2
3. a recombinant plasmid is characterized in that this recombinant plasmid is pET-29a (+) recombinant plasmid that contains fenoxapropPethyl hydrolysis esterase gene claimed in claim 1.
4. the genetic engineering bacterium that contains fenoxapropPethyl hydrolysis esterase gene claimed in claim 1
E.coliBL21 (DE3).
5. the construction process of genetic engineering bacterium claimed in claim 4 is characterized in that: pET-29a (+) recombinant plasmid transformed that contains fenoxapropPethyl hydrolysis esterase gene claimed in claim 3 is arrived the expressive host bacterium
E.coliBL21 (DE3) obtains genetic engineering bacterium, again the genetic engineering bacterium that obtains is transferred to the flat board that contains 200 mg/l fenoxapropPethyls, 50 mg/l kantlex and 24 mg/l IPTG, behind 37 ℃ of cultivation 16 h, picking has the positive transformant of hydrolysis, after the sequence verification gene order is errorless, preserve.
6. the described fenoxapropPethyl hydrolysis of claim 1 esterase gene is used in the genetically engineered aspect the fragrant phenoxy phenoxy propionic acid weedicide of hydrolysis, and described fragrant phenoxy phenoxy propionic acid weedicide is fenoxapropPethyl, quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl.
7. the application of the described fenoxapropPethyl ester hydrolysis of claim 2 zymoprotein aspect the fragrant phenoxy phenoxy propionic acid weedicide of hydrolysis, described fragrant phenoxy phenoxy propionic acid weedicide is fenoxapropPethyl, quizalofopPethyl, clodinafop-propargyl, cyhalofop-butyl.
8. the application of the described fenoxapropPethyl hydrolysis of claim 1 esterase gene in the hydrolysis triglyceride, described triglyceride is vanay, tributyrin or tricaproin.
9. the application of the described fenoxapropPethyl hydrolysis of claim 1 esterase gene in the hydrolysis p-nitrophenyl ester, described p-nitrophenyl ester is acetic acid p-nitrophenyl acetate, butyric acid p-nitrophenyl ester, caproic acid p-nitrophenyl ester, sad p-nitrophenyl ester, capric acid p-nitrophenyl ester, lauric acid p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester, palmitinic acid p-nitrophenyl ester.
10. application according to claim 9 is characterized in that described p-nitrophenyl ester is the butyric acid p-nitrophenyl ester.
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