CN102643841A - 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 98
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 82
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 81
- 108090000371 Esterases Proteins 0.000 title claims abstract description 64
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 title claims abstract description 13
- -1 p-nitrophenyl ester Chemical class 0.000 claims abstract description 32
- 239000013612 plasmid Substances 0.000 claims abstract description 17
- 238000010353 genetic engineering Methods 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 241000588724 Escherichia coli Species 0.000 claims description 15
- SXERGJJQSKIUIC-UHFFFAOYSA-N 2-Phenoxypropionic acid Chemical class OC(=O)C(C)OC1=CC=CC=C1 SXERGJJQSKIUIC-UHFFFAOYSA-N 0.000 claims description 10
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 claims description 10
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 10
- 150000002148 esters Chemical class 0.000 claims description 9
- 244000025254 Cannabis sativa Species 0.000 claims description 8
- 239000005502 Cyhalofop-butyl Substances 0.000 claims description 8
- TYIYMOAHACZAMQ-CQSZACIVSA-N Cyhalofop-butyl Chemical group C1=CC(O[C@H](C)C(=O)OCCCC)=CC=C1OC1=CC=C(C#N)C=C1F TYIYMOAHACZAMQ-CQSZACIVSA-N 0.000 claims description 8
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- OSUHJPCHFDQAIT-GFCCVEGCSA-N quizalofop-P-ethyl Chemical group C1=CC(O[C@H](C)C(=O)OCC)=CC=C1OC1=CN=C(C=C(Cl)C=C2)C2=N1 OSUHJPCHFDQAIT-GFCCVEGCSA-N 0.000 claims description 8
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
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] ethyl propionate be 1978 by German Hoechst (existing merger in German Bayer) company; A kind of fragrant oxygen propionic acid class 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 wheatland and prevent and kill off multiple gramineous weedss such as ild avena sativa, amur foxtail, also can prevent and kill off the annual and perennial gramineous weeds in beans, beet, cotton, flax, peanut and vegetables field.At present in the world and China use very wide.But big quantity research shows that fenoxapropPethyl shows as high toxicity to hydrobiont.
The degraded of environmental pollutant mainly depends on action of microorganisms in the soil, and mikrobe is mainly accomplished by intracellular enzyme the degraded of material.Mikrobe is the important source of various enzymes.Through from environment, separating microbes producing cellulase, utilize molecule clone technology from microbes producing cellulase, to clone and produce the enzyme gene, again it is connected with suitable carriers and changes corresponding host cell over to, can carry out the great expression of enzyme.At present, produce enzyme, become the leading of industrial enzymes through the genetic engineering technique means.
Esterase (esterase) broadly is meant the general name of one type of lytic enzyme with catalytic hydrolysis ester bond ability; And usually said esterase Procaine esterase (carboxylesterase often; E.C.3.1.1.1), mainly be used for hydrolysed fat acid family and aromatic series Ester.Triglyceride class and α-Nai Zhi and p-nitrophenyl Ester all are the specific substrates of esterase.Because esterase has very high activity,, utilize its hydrolysis reaction, ester conversion and ester synthesis reaction can be widely used in medicine, chemical industry, food, the energy and environmental protection field as biological catalyst.
Summary of the invention
The objective of the invention is to produce highly toxic problem to hydrobiont, a kind of fenoxapropPethyl hydrolysis esterase gene is provided to fenoxapropPethyl.
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 prosthetic, food-processing, medicine, washing of environment, not only can solve the problem of environmental pollution of fenoxapropPethyl, can also obtain considerable economic.
The object of the invention can be realized through following technical scheme:
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 is obtained is transferred to the flat board that contains 200mg/l fenoxapropPethyl, 50mg/l kantlex and 24mg/l IPTG; Behind 37 ℃ of cultivation 16h; Picking has the positive transformant of transparent hydrolysis circle; After the sequence verification gene order is errorless, preserve.
The genetic engineering application of said fenoxapropPethyl hydrolysis esterase gene aspect the hydrolysis of fragrant phenoxy propionic acid class weedicide.
Wherein, the preferred fenoxapropPethyl of described fragrant phenoxy propionic acid class weedicide, quizalofopPethyl, alkynes grass ester, cyhalofopbutyl.
The application of said fenoxapropPethyl hydrolysis esterase gene in the hydrolysis triglyceride, the preferred vanay of described triglyceride, tributyrin or tricaproin.
The application of said 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 99 MIN p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester, palmitinic acid p-nitrophenyl ester, further preferred butyric acid p-nitrophenyl ester.
The application of said fenoxapropPethyl ester hydrolysis zymoprotein aspect hydrolysis virtue phenoxy propionic acid class weedicide.
Wherein, described fragrant phenoxy propionic acid class weedicide is preferred fenoxapropPethyl, quizalofopPethyl, alkynes grass ester, cyhalofopbutyl.
The application of said fenoxapropPethyl ester hydrolysis zymoprotein in the hydrolysis triglyceride, the preferred vanay of described triglyceride, tributyrin or tricaproin.
The application of said 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 99 MIN 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 following:
The present invention with the fenoxapropPethyl be sole carbon source from soil separation screening to bacterial strain rhodococcus (Rhodococcus sp.) T1 of a high-efficiency degradation fenoxapropPethyl; And be its genomic library of material construction, and successfully from this library, clone fenoxapropPethyl hydrolysis esterase (one of key enzyme in the fenoxapropPethyl degradation process) gene with this bacterial strain.The product fenoxapropPethyl hydrolysis esterase of this genetic expression, the virtue of hydrolysis efficiently phenoxy propionic acid class weedicide fenoxapropPethyl, quizalofopPethyl, alkynes grass ester, cyhalofopbutyl etc.
2. utilize this gene constructed engineering strain can efficiently express fenoxapropPethyl hydrolysis esterase; This esterase can also act on other ester compounds such as triglyceride, p-nitrophenyl ester, and the zymin of production can be used for industries such as the biological prosthetic 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 efficiently expresses experimental program figure in E.coli BL21 (pET-29a (+)).
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
The clone of embodiment 1. fenoxapropPethyl hydrolysis esterase genes
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, enrichment continuously, and the concentration that progressively improves fenoxapropPethyl is to 200mg/l.After treating that the pregnant solution degradation effect is stable, adopt the gradient dilution flat band method, the pregnant solution of dilution is coated with 30 ℃ of cultivations containing on the MSM solid medium flat board of 100mg/l fenoxapropPethyl.After treating the single bacterium colony of dull and stereotyped upward 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 the degradation effect of single bacterium to 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 following well-growns.It is circular, moistening to cultivate visible oyster white of 24h or incarnadine, the heavy-gravity bacterium colony.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 to extract the total DNA of karyomit(e) of T1 (CGMCC No.5030), 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 pUC118 (BamH I/BAP) (and TaKaRa, Code:D3321, down with) connect after; Chemical conversion imports the E.coli DH5 α competent cell (TaKaRa for preparing; Code:D9057, down together), make up rhodococcus (Rhodococcus sp.) T1 genomic library.LB flat board through containing the 200mg/l fenoxapropPethyl is selected the positive colony that contains fenoxapropPethyl hydrolysis esterase gene.
1.2.1 the extraction of the total DNA of thalline
Adopt high salt method to extract the total DNA of karyomit(e) of rhodococcus (Rhodococcus sp.) T1 (CGMCC No.5030): the single colony inoculation of picking rhodococcus (Rhodococcus sp.) T1 (CGMCC No.5030) is in 3ml LB liquid nutrient medium; 30 ℃, 180rpm are cultured to OD600nm ≈ 1.0, the centrifugal collection thalline of 12000rpm; With the resuspended washing thalline of 1.0mL TE damping fluid (pH 8.0 for 10mmol/l TrisCl (pH8.0), 1mmol/l EDTA), 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, treat liquid clarification after, add the saturated NaCl solution of 340 μ l concuss; The centrifugal 10min of 12000rpm is transferred to supernatant in the aseptic clean eppendorf pipe, uses isopyknic phenol: chloroform: till the no white solid thing of primary isoamyl alcohol (25:24:1) extracting to interface clarification; Shift supernatant in another aseptic clean eppendort pipe, add the Virahol of 0.6 times of volume, place 0.5-1h deposit D NA for-20 ℃; The centrifugal 10min of 12000rpm removes behind the supernatant with 70% washing with alcohol 2 times, treats ethanol volatilization back adding 30 μ l sterilized water or TER; Place 4 ℃ of refrigerator overnight dissolvings, 4 ℃ of preservations of short-term ,-20 ℃ of long-term employings are frozen.
1.2.2 it is following 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, adds the Sau3AI enzyme switchback section of taking up of the total DNA of 6 μ l, and adds water to 8.5 μ l, makes again in 45 ℃ of 5min that heat that the annealed cohesive end unwinds, and mixture is cooled to 0 ℃ with 1 μ l pUC118.Add 1 μ l10 * T then
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.Transfer to fast and cool off 1 ~ 2min in the ice bath, in every pipe, add 800 μ l liquid LB substratum, 37 ℃ of shaking table 80-90rpm incubation 45min, recovery cell.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 board is inverted in 37 ℃ of incubators and cultivates, and occurs bacterium colony behind the 12-16h, selects to have clone's of hydrolysis circle; Be positive colony that contains fenoxapropPethyl hydrolysis esterase gene fragment, simultaneously positive colony numbered.Choose single colony inoculation in 3ml LB liquid tube, wait to grow upgrading grain checking behind the bacteria suspension.
1.4 positive colony inserts segmental order-checking and analysis
Positive colony inserts segmental order-checking and entrusts the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd to accomplish.Sequencing primer be pUC118 (Auele Specific Primer on the carrier of BamH I/BAP), and according to two ends sequencing result design primer, logical until whole insertion fragment is surveyed.
The sequence that order-checking is accomplished is compared in ncbi database, and utilize ORF finder instrument to identify and insert the contained ORF of 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) has the NdeI restriction enzyme site, reverse primer R1:5 '-
CTCGAGTCAACTCAAAGCGTCGTAGGC-3 ' (SEQ ID NO.4) has an XhoI restriction enzyme site, and the full gene of fenoxapropPethyl hydrolysis esterase is used to increase.A pair of in addition primers F 2:5 '-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 to make 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; Be template for the first time, utilize primers F 1 and R2 to increase comprising the fragment of XhoI restriction enzyme site in the fenoxapropPethyl hydrolysis esterase gene in interior left side with the positive colony plasmid that contains fenoxapropPethyl hydrolysis esterase gene through sequence verification in 1.4.Be that to utilize primers F 2 and R1 be that primer increases to comprising the fragment of XhoI restriction enzyme site on interior right side in the fenoxapropPethyl hydrolysis esterase gene to template for the second time with the positive colony plasmid.Twice amplification PCR products is template for the third time, utilizes primers F 1 and R1 to increase, and obtains the complete genome of fenoxapropPethyl hydrolysis esterase.The 4th time is template with amplified production for the third time, utilizes the Taq enzyme that amplified fragments is added the A tail.
Amplification for the first time:
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.
Amplification for the second time: change amplimer into F2 and R1, all the other increase identical with the first time.
Amplification for the third time:
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 that adds the A tail through PCR and pMD 19-T Vector (TaKaRa Code:D102A) 3:1 in molar ratio mix, and are connecting under the liquid effect, and 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 (with reference to 1.3) of positive colony
1.8 the extraction of the sub-plasmid of positive colony and order-checking
In 1.7, screen the positive colony overnight cultures in containing the LB substratum of penbritin that obtains, the centrifugal 10min of 12000rpm collects thalline, utilizes plasmid extraction kit to extract plasmid, serves the order-checking of the prompt basic biological ltd in the extra large English Weihe River.
Embodiment 2. fenoxapropPethyl hydrolysis esterase genes efficiently expressing 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) is with 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 transparent hydrolysis circle, 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.Collect thalline with phosphoric acid buffer (pH8.0) resuspended after, with supersound process fragmentation somatic cells, the centrifugal 15min of 20000g, gained supernatant are fenoxapropPethyl hydrolysis esterase crude enzyme liquid.Get 10 μ l fenoxapropPethyl hydrolysis esterase crude enzyme liquids 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 performance liquid chromatography.The enzyme unit definition of living is that PM produces 1 μ mol fenoxapropPethyl acid and is a unit; The ratio vigor that records thick enzyme is 31.83 μ mol/min/mg, and promptly this thick enzyme of 1mg is that substrate reacts PM and can obtain 31.83 μ mol fenoxapropPethyls acid with the fenoxapropPethyl under optimum condition.After be 2995.03 μ mol/min/mg with this enzyme through the Ni-NTA affinitive layer purification and through recording the ratio vigor of this enzyme when being substrate behind the ultrafiltration and concentration with the fenoxapropPethyl, promptly the pure enzyme of 1mg PM when fenoxapropPethyl is substrate produces the acid of 2995.03 μ mol fenoxapropPethyls.This result shows that the ratio enzyme activity after purifying concentrates is about 94 times of thick enzyme.
Embodiment 3 fenoxapropPethyl hydrolysis esterases are 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 0.02 mol/l vanay, tributyrin, tricaproin, capryiin and caprin respectively; PH7.5) in; React 10min down at 50 ℃; Add two 1% phenolphthalein solutions again after adding 10ml ethanol termination reaction, to lightpink, write down the NaOH consumption with 0.01mol/l NaOH titration.The enzyme unit definition of living is that PM 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 capryiin and caprin.
Embodiment 4 fenoxapropPethyl hydrolysis esterases are 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 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 99 MIN p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester and palmitinic acid p-nitrophenyl ester respectively; PH8.0) in; 50 ℃ of reactions down; Dilute 10 times behind the 5min immediately, measure OD
410nmThe enzyme unit definition of living is that PM 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 99 MIN 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 fenoxapropPethyl hydrolysis esterases are to the hydrolysis of other fragrant phenoxy propionic acid class weedicides
With the fenoxapropPethyl hydrolysis esterase (preparation method is with embodiment 2) of 1.47 μ g purifying join 4ml contain respectively in the phosphoric acid buffer of 100mg/l quizalofopPethyl, alkynes grass ester, cyhalofopbutyl (0.02mol/l, pH8.0), at 50 ℃ of reactions down, 5min.Add 10%HCl and regulate pH to 2.0, add the extracting of equal-volume methylene dichloride.The phase of anhydrating adds SODIUM SULPHATE ANHYDROUS 99PCT 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, alkynes grass ester, cyhalofopbutyl with performance liquid chromatography.The enzyme unit definition of living is that the PM corresponding substrate of 1 μ mol of degrading is a unit, records pure enzyme the ratio vigor of quizalofopPethyl, alkynes grass ester and cyhalofopbutyl 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. the fenoxapropPethyl ester hydrolysis zymoprotein of the described fenoxapropPethyl hydrolysis of claim 1 esterase coded by said gene, 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 the described fenoxapropPethyl hydrolysis of claim 1 esterase gene.
4. contain the genetic engineering bacterium E.coli BL21 (DE3) that right requires 1 described fenoxapropPethyl hydrolysis esterase gene.
5. the construction process of the described genetic engineering bacterium E.coli of claim 4 BL21 (DE3); It is characterized in that: described pET-29a (+) recombinant plasmid transformed that contains fenoxapropPethyl hydrolysis esterase gene of claim 3 is obtained recombinant microorganism E.coli BL21 (DE3) to expressive host bacterium E.coliBL21 (DE3); Again the recombinant microorganism E.coli BL21 (DE3) that is obtained is transferred to the flat board that contains 200mg/l fenoxapropPethyl, 50mg/l kantlex and 24mg/l IPTG; Behind 37 ℃ of cultivation 16h; Picking has the positive transformant of transparent hydrolysis circle, after the sequence verification gene order is errorless, preserves.
6. the genetic engineering application of the said fenoxapropPethyl hydrolysis of claim 1 esterase gene aspect hydrolysis virtue phenoxy propionic acid class weedicide.
7. application according to claim 6 is characterized in that described fragrant phenoxy propionic acid class weedicide is fenoxapropPethyl, quizalofopPethyl, alkynes grass ester, cyhalofopbutyl.
8. the application of the said fenoxapropPethyl ester hydrolysis of claim 2 zymoprotein aspect hydrolysis virtue phenoxy propionic acid class weedicide, the preferred fenoxapropPethyl of described fragrant phenoxy propionic acid class weedicide, quizalofopPethyl, alkynes grass ester, cyhalofopbutyl.
9. the application of the said fenoxapropPethyl hydrolysis of claim 1 esterase gene in the hydrolysis triglyceride, the preferred vanay of described triglyceride, tributyrin or tricaproin.
10. the application of the said fenoxapropPethyl hydrolysis of claim 1 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 99 MIN p-nitrophenyl ester, tetradecanoic acid p-nitrophenyl ester, palmitinic acid p-nitrophenyl ester, further preferred butyric acid p-nitrophenyl ester.
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| CN107012132A (en) * | 2016-10-12 | 2017-08-04 | 南京工业大学 | Aryloxy phenoxy propionic acid herbicide hydrolysis esterase and application thereof |
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