CN107022556A - The method of one kind bacillus subtilis engineering bacteria 1,2,3 trichloropropanes of degraded - Google Patents

The method of one kind bacillus subtilis engineering bacteria 1,2,3 trichloropropanes of degraded Download PDF

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CN107022556A
CN107022556A CN201710198807.4A CN201710198807A CN107022556A CN 107022556 A CN107022556 A CN 107022556A CN 201710198807 A CN201710198807 A CN 201710198807A CN 107022556 A CN107022556 A CN 107022556A
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刘玉焕
孔伟
任广辉
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National Sun Yat Sen University
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Abstract

The invention discloses one kind bacillus subtilis engineering bacteria 1,2,3 trichloropropanes of degraded（TCP）Method.Synthesis first can degrade alkyl halide dehalogenation enzyme gene, halide alcohol dehalogenase gene and the epoxide hydrolase gene of 1,2,3 trichloropropanes and its intermediate product, and the amino acid sequence of gene order and its codase is respectively as shown in SEQ ID NO.1~6；Then the bacillus subtilis integration vector for including said gene is built, carrier converts bacillus subtilis, the method knocked out by gene ectopic integration and resistant gene, said gene is integrated into Bacillus subtilis genes group, acquisition can be with the bacillus subtilis engineering bacteria of secreting, expressing alkyl halide dehalogenase, halide alcohol dehalogenase and epoxide hydrolase.The engineering bacteria all has good degradation to TCP and its intermediate product, and the degradable of TCP can be achieved, with degradation efficiency is high, strong TCP tolerances and the features such as inheritance stability, and application prospect is good.

Description

The method of one kind bacillus subtilis engineering bacteria 1,2,3- trichloropropanes of degraded
Technical field
The invention belongs to biological technical field.More particularly, to one kind bacillus subtilis engineering bacteria degraded 1,2, The method of 3- trichloropropanes.
Background technology
1,2,3- trichloropropane（TCP）It is a kind of artificial synthesized chloro organic cpd, is manufacture paint and soil The important source material of earth insecticide, is widely used in industry and agricultural production.But TCP has the characteristics of high toxicity and difficult degradation, The a large amount of accumulation for causing the material in soil and groundwater are widely used, human health and ecological environment are caused huge Threat.
Degraded TCP method has at present：Physics, chemistry and biological method.Physical includes vacuum filtration and activated carbon is inhaled It is attached.But TCP is volatile, difficult absorption, so Physical can not effectively solve TCP pollution problems.Chemical method mainly utilizes Strong oxdiative Agent and strong reductant processing TCP, with technique it is simple the characteristics of.But the use of strong oxidizer and strong reductant can heavy damage life State environment.Compared with physics and chemical method, biological degradation method has the advantages that cost is low and can be degradable by harmful substance, Turn into the effective ways of degraded environmental contaminants.At present, there is research and utilization immobilised enzymes（Alkyl halide dehalogenase, halide alcohol dehalogenase And epoxide hydrolase）External TCP degradation pathways are built, TCP is changed into glycerine.But there is manufacturing process and answer in immobilised enzymes The problem of miscellaneous, production cost is high and is difficult to recycling, particularly in the TCP pollution environment of low concentration, immobilised enzymes has Obvious limitation.In addition, some research and establishments engineering bacteria of can degrade TCP and its intermediate product.But the bacterial strain built is deposited The problem of TCP poor resistances, Genomic instability and low degradation efficiency.
The content of the invention
The purpose of the present invention is that the defect and deficiency for overcoming above-mentioned existing TCP degradation techniques use withered grass gemma there is provided one kind The method of bacillus engineering bacteria 1,2,3- trichloropropanes of degraded.
It is a further object of the present invention to provide a kind of bacillus subtilis engineering bacteria of 1,2,3- trichloropropanes of degraded.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of alkyl halide dehalogenation enzyme gene（dhaA31）, its nucleotide sequence is as shown in SEQ ID NO.1.
A kind of alkyl halide dehalogenase（haloalkanedehalogenase, DhaA31）, its amino acid sequence such as SEQ ID Shown in NO.2.
A kind of halide alcohol dehalogenase gene（hheC）, its nucleotide sequence is as shown in SEQ ID NO.3.
A kind of halide alcohol dehalogenase（halohydrindehalogenase, HheC）, its amino acid sequence such as SEQ ID Shown in NO.4.
A kind of epoxide hydrolase gene（echA）, its nucleotide sequence is as shown in SEQ ID NO.5.
A kind of epoxide hydrolase（epoxide hydrolase, EchA）, its amino acid sequence such as SEQ ID NO.6 It is shown.
The bacillus subtilis engineering bacteria of one kind 1,2,3- trichloropropanes of degraded, construction method is：Synthesis can degrade first 1,2,3- trichloropropane（TCP）And its intermediate product（2,3- bis- trimethylewne chlorohydrin 3-s and epoxychloropropane）Alkyl halide dehalogenase （DhaA31）Gene, halide alcohol dehalogenase（HheC）Gene and epoxide hydrolase（EchA）Gene, sequence is respectively such as SEQ ID Shown in NO.1, SEQ ID NO.3, SEQ ID NO.5；Then the bacillus subtilis integration vector for including said gene is built, Carrier converts bacillus subtilis, the method knocked out by gene ectopic integration and resistant gene, said gene is integrated into withered In careless bacillus gene group.Alkyl halide dehalogenase, the halogen of gained bacillus subtilis engineering bacteria secreting, expressing said gene coding Alcohol dehalogenase and epoxide hydrolase.
Specifically, it is preferable that the construction method of the bacillus subtilis engineering bacteria comprises the following steps：
（1）Gene chemical synthesis：Alkyl halide dehalogenase is optimized according to the codon preference of bacillus subtilis（DhaA31）, halogenohydrin dehalogenation Enzyme（HheC）And epoxide hydrolase（EchA）Gene order, and synthesize alkyl halide dehalogenation enzyme genedhaA31st, halide alcohol dehalogenase GenehheCAnd epoxide hydrolase geneechA；
（2）Using Pgrac promoters and PhoD signal peptide sequences, build include respectivelydhaA31 genes,hheCGene andechA The bacillus subtilis integration vector of gene；
（3）By step（2）Integration vector conversion bacillus subtilis, pass through the side that gene ectopic integration and resistant gene are knocked out Method, willdhaA31 genes,hheCGene andechAGene is integrated into Bacillus subtilis genes group successively, and structure obtains dividing Secrete the bacillus subtilis engineering bacteria of expression alkyl halide dehalogenase, halide alcohol dehalogenase and epoxide hydrolase.
More specifically, being used as a kind of selectable preferred scheme, the construction method bag of the bacillus subtilis engineering bacteria Include following steps：
S1. the related components needed for enzyme gene and structure integration vector are prepared
S11. alkyl halide dehalogenase is synthesized（DhaA31）Gene, halide alcohol dehalogenase（HheC）Gene and epoxide hydrolase（EchA） Gene, sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5；
S12. target gene alkyl halide dehalogenation enzyme gene, halide alcohol dehalogenase gene and epoxide hydrolase gene are inserted respectively into In plasmid pMD18-T, structure obtains recombinant plasmid pMD18T-dhaA31、 pMD18T-hheCAnd pMD18T-echA；
S13.PCR amplification obtain build integration vector needed for promoter and signal peptide sequence
S131. promoter sequence is expanded
Using plasmid pHT43 as carrier, primer is utilizedPgrac- fw and Pgrac-rv enter performing PCR amplification, obtain promoterPgracSequence Row；
Primer Pgrac-fw sequence is as shown in SEQ ID NO.7, and primer Pgrac-rv sequence is as shown in SEQ ID NO.8；
S132. amplified signal peptide sequence
Expanded and obtained from Bacillus subtilis genes group DNA using primer PhoD-fw and PhoD-rvphoDSignal peptide sequence；
Primer PhoD-fw sequence is as shown in SEQ ID NO.9, and primer PhoD-rv sequence is as shown in SEQ ID NO.10；
S133. the promoter of amplification and signal peptide sequence are connected with T4DNA ligases, structure obtains including promoter and signal The DNA sequence dna component of peptide（Pgrac-phoDcassette）, as shown in SEQ ID NO.13；
S2. bacillus subtilis integration vector is built
S21. construction recombination plasmid pDGIEF-PSphoddhaA31
Encoding plasmids pDGIEF gene order is downloaded from GenBank Data Base databases（Serial No. DQ358863.1）, plasmid pDGIEF is synthesized in Genaray Company（Shanghai Generay Biotech Co., Ltd）, as host's popularity plasmid pDGIEF；
With recombinant plasmid pMD18T-dhaA31 be template, using primerdhaA31-fw/dhaA31-rvEnter performing PCR amplification acquisition Foreign genedhaA31；Amplified production is inserted into plasmid pDGIEF's after double digestion is handledSalI/NotI site, builds Obtain recombinant plasmid pDGIEF-dhaA31；
WillPgrac-Sphod Recombinant plasmid pDGIEF- is inserted into after cassette double digestionsdhaA31NheI/SalI site, Structure obtains recombinant plasmid pDGIEF-PSphoddhaA31；
Wherein, primer dhaA31-fw sequence is as shown in SEQ ID NO.14, primer dhaA31-rvSequence such as SEQ ID Shown in NO.15；
S22. construction recombination plasmid pIEFVPR-PSphoDhheC
With pMD18T-hheCFor template, primer is usedhheC-fw/hheC- rv enters performing PCR amplification and obtains foreign genehheC；Expand Increase production thing after double digestion is handled, be inserted intovprGene loci integrated plasmid pIEFVPR'sSalI/NotI site, builds To recombinant plasmid pIEFVPR-hheC；
WillPgrac-Sphod Plasmid pIEFVPR- is inserted into after the processing of cassette double digestionshheC'sNheI/SalI site, Structure obtains recombinant plasmid pIEFVPR-PSphoDhheC；
Wherein, primerhheC- fw sequence is as shown in SEQ ID NO.24, primerhheC- rv sequence such as SEQ ID NO.25 It is shown；
S23. construction recombination plasmid pIEFEPR-PSphoDechA
With pMD18T-echAFor template, primer is usedechA-fw/echA-Rv enters performing PCR amplification and obtains foreign geneechA；Expand Increase production thing after double digestion is handled, be inserted intoeprGene loci integrated plasmid pIEFEPR'sSalI/NotI site, builds To recombinant plasmid pIEFEPR-echA；WillPgrac-SphodCassette is inserted into plasmid pIEFEPR- after being handled through double digestionechA'sNheI/SalI site, structure obtains recombinant plasmid pIEFEPR-PSphoDechA；
Wherein, primerechA- fw sequence is as shown in SEQ ID NO.34, primerechA-Rv sequence such as SEQ ID NO.35 It is shown；
The PCR programs for expanding foreign gene using primer are as follows：98 DEG C, 10 min；98 DEG C, 10 s；55 DEG C, 10 s；72 DEG C, 15 s carry out 30 circulations；72 DEG C, 3 min；
S3. bacillus subtilis engineering bacteria is built
S31. recombinant plasmid pDGIEF-PSphoddhaA31 conversion competent escherichia coli cells, extract recombinant plasmid, linearisation With host genome double crossing over homologous recombination occurs for recombinant plasmid transformed bacillus subtilis bacterium competence cell afterwards, recombinant plasmid, Target gene is integrated into genomeamyeSite；Positive colony is selected from the LB flat boards containing spectinomycin, is inoculated with Into LB culture mediums, 37 DEG C, 200 rpm cultivate 24 h, and culture is diluted, the solid LB containing 0.1 mM IPTG is coated on Flat board, filters out IPTGsAnd spcrSingle bacterium colony, as contain foreign genedhaA31 engineering bacteria, is named asB. subtilis 168-1；
S32. same method is used, by halide alcohol dehalogenase gene（hheC）And epoxide hydrolase gene（echA）It is whole successively Close engineering bacteriaB. subtilis 168-1 genomesvprWitheprSite, final build is obtained containing foreign genedhaA31、hheCWithechABacillus subtilis engineering bacteria.
In addition, present invention also offers a kind of method for 1,2,3- trichloropropanes of being degraded with bacillus subtilis engineering bacteria, It is to utilize above-mentioned bacillus subtilis engineering bacteria 1,2,3- trichloropropanes of degraded.
The bacillus subtilis engineering bacteria that the present invention is built is resistant to 8mM 1,2,3- trichloropropanes（TCP）, and Under the conditions of 30~37 DEG C, in basic salt culture medium（MMY）Middle culture 20~30 days, engineering bacteria can be degradable by 2 mM TCP.
Therefore, bacillus subtilis engineering bacteria is in 1,2,3- trichloropropanes and its intermediate product of degrading（2,3- bis- chloro- 1- third Alcohol and epoxychloropropane）Application, and preparing 1,2,3- trichloropropanes, the trimethylewne chlorohydrin 3-s of 2,3- bis- and/or epoxychloropropane Degraded preparation in terms of application, also within protection scope of the present invention.
The present invention builds dehalogenase containing alkyl halide by Protocols in Molecular Biology（DhaA31）Gene, halide alcohol dehalogenase（HheC） Gene and epoxide hydrolase（EchA）The bacillus subtilis integration vector of gene；Above-mentioned carrier is converted into bacillus subtilis Bacterium；The method knocked out by gene ectopic integration and resistant gene, said gene is integrated into respectively the base of bacillus subtilis 168 Because of group；Alkyl halide dehalogenase, halide alcohol dehalogenase and the epoxide hydrolase secreted by bacillus subtilis engineering bacteria, build 1, 2,3- trichloropropane degradation pathways, glycerine is changed into by TCP, finally realizes the permineralization of 1,2,3- trichloropropanes.
The invention has the advantages that：
The bacillus subtilis engineering bacteria that the present invention is built being capable of secreting, expressing degraded 1,2,3- trichloropropanes（TCP）And its it is middle Alkyl halide dehalogenase, halide alcohol dehalogenase and the epoxide hydrolase of product, to 1,2,3- trichloropropane（TCP）Better tolerance is right TCP and its intermediate product have good degradation effect and efficiency, and the degradable of TCP can be achieved, with degradation efficiency it is high, TCP tolerances are strong and are the current degraded maximally effective genetic engineering bacteriums of TCP the features such as inheritance stability.
Brief description of the drawings
Fig. 1：Bacillus subtilis integration vector；P：Pgrac promoters；S：PhoD signal peptides；dhaA31：Alkyl halide dehalogenase Mutant gene；hheC：Halide alcohol dehalogenase gene；echA：Epoxide hydrolase gene.amye-front：amyeOn gene Swim homology arm；amye-back：amyeDownstream of gene homology arm；vpr-front：vprUpstream region of gene homology arm；vpr-back：vprDownstream of gene homology arm；epr-front：eprUpstream region of gene homology arm；epr-back：eprDownstream of gene homology arm.
Fig. 2：Alkyl halide dehalogenation enzyme gene（dhaA31）, halide alcohol dehalogenase gene（hheC）And epoxide hydrolase gene （echA）It is incorporated into Bacillus subtilis genes groupamye、vprWitheprSite.
Fig. 3：Alkyl halide dehalogenation enzyme gene is verified by PCR amplifications and agarose gel electrophoresis technology（dhaA31）, halogenohydrin take off Halogen enzyme gene（hheC）And epoxide hydrolase gene（echA）It is incorporated into Bacillus subtilis genes groupamye、vprWithepr Site；M：Marker；Line 1：Control groupamyeLocus gene fragment；Line 2：Bacillus subtilis engineering bacteriaamyeSite Genetic fragment；Line 3：Control groupvprLocus gene fragment；Line 4.：Bacillus subtilis engineering bacteriavprLocus gene piece Section；Line 5：Control groupeprLocus gene fragment；Line 6：Bacillus subtilis engineering bacteriaeprLocus gene fragment.
Fig. 4：Alkyl halide dehalogenase（DhaA31）, halide alcohol dehalogenase（HheC）And epoxide hydrolase（EchA）In withered grass bud Expression and secretion in spore bacillus engineering bacteria（Foreign protein in bacillus subtilis recombinant base supernatant）；M： Marker；Line1：Alkyl halide dehalogenase；Line 2：Halide alcohol dehalogenase；Line 3：Epoxide hydrolase.
Fig. 5：Bacillus subtilis engineering bacteriaB. subtilis Tolerances of the 168-3 to 1,2,3- trichloropropane.
Fig. 6：Degradation of the bacillus subtilis recombinant supernatant crude enzyme liquid to 1,2,3- trichloropropane.
Fig. 7：Bacillus subtilis Engineering Bureau bacterium is to 1,2,3- trichloropropane, the trimethylewne chlorohydrin 3-s of 2,3- bis- and epoxychloropropane Degradation；A is degradation of the engineering bacteria to TCP；B is degradation of the engineering bacteria to DCP；C is engineering bacteria to ECH's Degradation.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Embodiment 1 obtains the related components needed for enzyme gene and structure integration vector
1st, optimized according to bacillus subtilis codon preference, have devised alkyl halide dehalogenation enzyme mutant gene（dhaA31, core Acid sequence is as shown in SEQ ID NO.1）, halide alcohol dehalogenase gene（hheC, nucleotide sequence is as shown in SEQ ID NO.3）And epoxy Compound hydrolase gene（echA, nucleotide sequence is as shown in SEQ ID NO.5）.
Correspondence coding obtains alkyl halide dehalogenase（haloalkanedehalogenase, DhaA31）Amino acid sequence such as SEQ ID NO.2 are shown, halide alcohol dehalogenase（halohydrindehalogenase, HheC）Amino acid sequence such as SEQ ID NO.4 institutes Show, epoxide hydrolase（epoxide hydrolase, EchA）Amino acid sequence is as shown in SEQ ID NO.6.
2nd, the above-mentioned purpose gene of synthesis is inserted into plasmid pMD18-T, construction recombination plasmid pMD18T-dhaA31、 pMD18T-hheCAnd pMD18T-echA。
3rd, PCR amplification obtain build integration vector needed for promoter and signal peptide sequence
（1）Expand promoter sequence
With plasmid pHT43 (VT2007, MiaoLingBio, Guangzhou, China) for carrier, primer is utilizedPgrac-fw Enter performing PCR amplification with Pgrac-rv, Pgrac promoter sequences are obtained, as shown in SEQ ID NO.11.
Primer Pgrac-fw（Sequence is as shown in SEQ ID NO.7）：
5’-ACACTAGCTAGCAGCTATTGTAACATAATCGGTACGGG-3’（Underscore part isNheI restriction enzyme sites, 5 ' 6, end is digestion protection base）；
Primer Pgrac-rv（Sequence is as shown in SEQ ID NO.8）：
5’-TAACGCGGATCCTTCCTCCTTTAATTGGG-3’（Underscore part isBamHIRestriction enzyme site, 5 ' 6, ends are enzymes Cut protection base）.
（2）Amplified signal peptide sequence
Expanded and obtained from Bacillus subtilis genes group DNA using primer PhoD-fw and PhoD-rvphoDSignal peptide sequence, As shown in SEQ ID NO.12.
Primer PhoD-fw（Sequence is as shown in SEQ ID NO.9）：
5’- TAACGCGGATCCATGGCATACGACAGTC-3’（Underscore part isBamHIRestriction enzyme site, 5 ' 6, ends are enzymes Cut protection base）；
Primer PhoD-rv（Sequence is as shown in SEQ ID NO.10）：
5’-GCGCCGGTCGACAGCATTTACTTCAAAGGC-3’（Underscore part isSalIRestriction enzyme site, 5 ' 6, ends are enzymes Cut protection base）.
（3）The promoter of amplification and signal peptide sequence are connected with T4DNA ligases, structure obtains including promoter and letter The DNA sequence dna component of number peptide（Pgrac-phoDcassette）, as shown in SEQ ID NO.13.
Embodiment 2 builds bacillus subtilis integration vector
1st, construction recombination plasmid pDGIEF-PSphoddhaA31
（1）Encoding plasmids pDGIEF gene order is downloaded from GenBank Data Base databases（Serial No. DQ358863.1）, plasmid pDGIEF is synthesized in Genaray Company（Shanghai Generay Biotech Co., Ltd）, as host's popularity plasmid pDGIEF.
（2）With recombinant plasmid pMD18T-dhaA31 be template, using primerdhaA31-fw/dhaA31-rvEnter performing PCR Amplification obtains foreign genedhaA31；Amplified production is inserted into plasmid pDGIEF's after double digestion is handledSalI/NotI Point, structure obtains recombinant plasmid pDGIEF-dhaA31。
PCR programs are as follows：98 DEG C, 10 min；98 DEG C, 10 s；55 DEG C, 10 s；72 DEG C, 15 s carry out 30 and followed Ring；72 DEG C, 3 min.
Primer dhaA31-fw（Sequence is as shown in SEQ ID NO.14）：
5’-ATCCGCGTCGACATGTCAGAAATTGGCACAGGCTTTCCGTTTG-3’（Underscore part is SalI digestions position Point）；
Primer dhaA31-rv（Sequence is as shown in SEQ ID NO.15）：
5’-TATTAGCGGCCGCTTAATGATGGTGATGATGATGCAGTGCCGGC-3’（Underscore part isNotI digestions position Point）.
（3）Using primer Pgrac-fw/PhoD-rv fromPgrac-SphoD PCR amplifications are obtained on DNA sequence dna componentPgrac-Sphod Cassette, is inserted into recombinant plasmid pDGIEF- after being handled through double digestiondhaA31NheI/SalI Point, structure obtains recombinant plasmid pDGIEF-PSphoddhaA31（As shown in Figure 1）.
2nd, construction recombination plasmid pIEFVPR-PSphoDhheC
（1）Construction recombination plasmid pIEFVPR1（Construction of recombinant plasmid method refers to Zhan et al, 2006）：
1）Using plasmid pDGIEF as carrier, using primerMazE- f andAmpOn-rv amplification plasmid vectorsmazE-AmpSequence；
Wherein, primerMazE-fw（Sequence is as shown in SEQ ID NO.16）：
5’-TTGGCGCGCCCGATAACCAGAAGC-3’（Underscore part isAscI restriction enzyme sites）；
PrimerAmp-rv1（Sequence is as shown in SEQ ID NO.17）：
5’-CGTGGCCAGGCGCGCCATGAGTATTCAAC-3’（Underscore part isAgeI restriction enzyme sites）.
2）Using on primer DR (up)-fw and DR (down)-rv amplification plasmid vectorsDR-LacI-mazF-spc-DRBase Because of module；
Primer DR (up)-fw（Sequence is as shown in SEQ ID NO.18）：
5’-GGACTAGTAAAATTGAAAAAATGGTGG-3’（Underscore part isSpeI restriction enzyme sites）；
Primer DR (down)-rv（Sequence is as shown in SEQ ID NO.19）：
5’-CGACGCGTGATCCCCCTATGCAAGGGTTTATTG-3’（Underscore part isMluI restriction enzyme sites）.
3）Using primervpr(front)-fw andvpr(front)-rv is expanded from Bacillus subtilis genes group DNAvprUpstream region of gene homology arm sequence (vpr-front)；
Primervpr(front)-fw（Sequence is as shown in SEQ ID NO.20）：
5’-CGTGGCCAGGATCATTCGCTTTCTGCTTG-3’（Underscore part isAgeI restriction enzyme sites）；
Primervpr(front)-rv（Sequence is as shown in SEQ ID NO.21）：
5’-GGACTAGTATCCGACTGTCCAGCCGTTCGG-3’（Underscore part isSpeI restriction enzyme sites）.
4）Using primervpr(back)-fw andvpr(back)-rv is expanded from Bacillus subtilis genes group DNAvpr Downstream of gene homology arm sequence（vpr-back）；
Primervpr(back)-fw（Sequence is as shown in SEQ ID NO.22）：
5’-CGACGCGTCAATAACAAAGAAGTTGAGC-3’（Underscore part isMluI restriction enzyme sites）；
Primervpr(back)-rv（Sequence is as shown in SEQ ID NO.23）：
5’-TTGGCGCGCCCGGTCAAAACCTGGCTTGAC-3’（Underscore part isAscI restriction enzyme sites）.
5）T is used after the DNA fragmentation of above-mentioned amplification is handled through restriction enzyme digestion4DNA ligase is attached, and is obtained To recombinant plasmid pIEFVPR1.
（2）With pMD18T-hheCFor template, primer is usedhheC-fw/hheC- rv enters performing PCR amplification, obtains external source base CausehheC.PCR response procedures are ibid.PIEFVPR1 is inserted into after amplified fragments are carried out into double digestion processing（vprGene position o'clock sharp Conjugative plasmid）'sSalI/NotI site, structure obtains recombinant plasmid pIEFVPR-hheC。
PrimerhheC-fw（Sequence is as shown in SEQ ID NO.24）：
5’-ATCCGCGTCGACATGTCTACAGCAATCGTTAC-3’（Underscore part isSalI restriction enzyme sites）；
PrimerhheC-rv（Sequence is as shown in SEQ ID NO.25）：
5’-TAATAGCGGCCGCTTAATGATGATGATGATGATGTTCC-3’（Underscore part isNotI restriction enzyme sites）.
（3）Using primer Pgrac-fw/PhoD-rv fromPgrac-SphoD PCR amplifications are obtained on DNA sequence dna componentPgrac-Sphod Cassette, is inserted into plasmid pIEFVPR- after being handled through double digestionhheC'sNheI/SalI site, builds Obtain recombinant plasmid pIEFVPR-PSphoDhheC（As shown in Figure 1）.
3rd, construction recombination plasmid pIEFEPR-PSphoDechA
（1）Construction recombination plasmid pIEFEPR2
1）Using pDGIEF as carrier, using primerMazE- fw andAmpOn-rv2 amplification plasmid vectorsmazE-AmpSequence；
PrimerMazE-fw（Sequence is as shown in SEQ ID NO.26）：
5’-TTGGCGCGCCCGATAACCAGAAGC-3’（Underscore part isAscI restriction enzyme sites）；
PrimerAmp-rv2（Sequence is as shown in SEQ ID NO.27）：
5’-CGACGCGTGGCGCGCCATGAGTATTCAAC-3’（Underscore part is MluI restriction enzyme sites）.
2）Using on primer DR (up)-fw2 and DR (down)-rv2 amplification plasmid vectorsDR-LacI-mazF-spc-DR Gene order component；
Primer DR (up)-fw2（Sequence is as shown in SEQ ID NO.28）：
5’-CAGGACGTCCAAAATTGAAAAAATGGTGG-3’（Underscore part isSbfI restriction enzyme sites）；
Primer DR (down)-rv2（Sequence is as shown in SEQ ID NO.29）：
5’-CGAGGCCTGATCCCCCTATGCAAGGGTTTATTG-3’（Underscore part isBspeI restriction enzyme sites）.
3）Using primerepr(front)-fw andepr(front)-rv is expanded from Bacillus subtilis genes group DNAeprUpstream region of gene homology arm sequence (epr-front)；
Primerepr(front)-fw（Sequence is as shown in SEQ ID NO.30）：
5’-CGACGCGTTGTATCAGTCACTCTG-3’（Underscore part isMluI restriction enzyme sites）；
Primerepr(front)-rv（Sequence is as shown in SEQ ID NO.31）：
5’-CAGGACGTCCTGGCTCCGATAATCCCTGCGAC-3’（Underscore part isSbfI restriction enzyme sites）.
4）Using primerepr(back)-fw andepr(back)-rv is expanded from Bacillus subtilis genes group DNAvpr Downstream of gene homology arm sequence（epr-back）；
Primerepr(back)-fw（Sequence is as shown in SEQ ID NO.32）：
5’-CGAGGCCTTTCAAAGTCTTCTCCAAGG-3’（Underscore part isBspeI restriction enzyme sites）；
Primerepr(back)-rv（Sequence is as shown in SEQ ID NO.33）：
5’-TTGGCGCGCCCGTCTTCAAATTGGTGCTCTTCAC-3’（Underscore part isAscI restriction enzyme sites）.
5）T is used after the DNA fragmentation of above-mentioned amplification is handled through digestion4DNA ligase is attached, and obtains recombinant plasmid pIEFEPR2。
（2）With pMD18T-echAFor template, using primerechA-fw/ echA-Rv is to foreign geneechAEnter performing PCR Amplification.PCR amplification programs are ibid.Amplified production is subjected to double digestion processing, pIEFEPR2 is inserted into（eprGene loci is integrated Plasmid）'sSalI/NotI site, structure obtains recombinant plasmid pIEFEPR-echA。
PrimerechA-fw（Sequence is as shown in SEQ ID NO.34）：
5’-ATCCGCGTCGACATGACGATTCGCCGCCCTGAAGATTTTAAAC-3’ （Underscore part isSalI digestions position Point）；
PrimerechA-rv（Sequence is as shown in SEQ ID NO.35）：
5’-TTATAGCGGCCGCTTAATGATGATGATGATGATGTCTGAATGC-3’（Underscore part isNotI digestions position Point）.
（3）Using primer Pgrac-fw/PhoD-rv fromPgrac-SphoD PCR amplifications are obtained on DNA sequence dna componentPgrac-Sphod Cassette, is inserted into plasmid pIEFEPR- after being handled through double digestionechA'sNheI/SalI site, builds Obtain recombinant plasmid pIEFEPR-PSphoDechA（As shown in Figure 1）.
Embodiment 3 builds bacillus subtilis engineering bacteria
1st, the bacillus subtilis integration vector of above-mentioned structure is converted into competent escherichia coli cell, extracts recombinant plasmid（Such as Shown in Fig. 2 and 3）：
（1）Recombinant plasmid pDGIEF-PSphoddhaA31 conversion competent escherichia coli cells, extract recombinant plasmid, by changing Conversion method is learned by recombinant plasmid pDGIEF-PSphoddhaAAfter 31 linearisations, bacillus subtilis bacterium competence cell, restructuring are converted With host genome double crossing over homologous recombination occurs for plasmid, and target gene is integrated into genomeamyeSite.From containing grand Positive colony is selected on the LB flat boards of mycin, is inoculated into 10 ml LB culture mediums, 37 DEG C, 200 rpm cultivate 24 h, Culture is diluted, the solid LB flat boards containing 0.1 mM IPTG is coated on, filters out IPTGsAnd spcrSingle bacterium colony, be Contain foreign genedhaA31 engineering bacteria, is named asB. subtilis 168-1。
（2）Using same method, by halide alcohol dehalogenase gene（hheC）And epoxide hydrolase gene（echA）According to It is secondary to be incorporated into engineering bacteriaB. subtilis 168-1 genomesvprWitheprSite, structure obtains engineering bacteriaB. subtilis 168-2, engineering bacteriaB. subtilis 168-3。
2nd, above-mentioned engineering bacteria is cultivated into 24 h in LB culture mediums, collects supernatant, concentrated with super filter tube.
Western Blot are analyzed, and as a result show to contain destination protein in supernatant, its molecular weight and the protein molecular of prediction Amount is in the same size（As shown in Figure 4）.The result proves that above-described 3 kinds of genes are expressed and secreted in bacillus subtilis To extracellular.
Application bacillus subtilis engineering bacteria 1,2, the 3- trichloropropanes of degraded of embodiment 4
1st, engineering bacteriaB. subtilis 168-3 incubated overnights in LB fluid nutrient mediums, then by 5000 g, 10 min from Heart processing, collects thalline, is washed with MMY culture mediums 2 times, and thalline is resuspended, the bacterium solution after resuspension is inoculated into containing 10 mM glucose 50 ml MMY culture mediums in, initial OD600=0.05, it is separately added into 1,2,3- trichloropropanes（TCP）, the trimethylewne chlorohydrin 3-s of 2,3- bis- （DCP）And epoxychloropropane（ECH）To final concentration of 2 mM.37 DEG C, cultivate under the conditions of 220 rpm.Between at a fixed time Every 1 ml reaction solutions of interior extraction, the acetone soln containing 0.05 mM n-hexyl alcohols is added（1：1）, examined by GC-MS chromatographs Chemical composition and concentration in test sample product.
2nd, the concentration of compound is tested and analyzed by GC-MS chromatographs.Sample adds after being extracted from reaction solution Enter the acetone soln containing 0.05 mM n-hexyl alcohols（1：1）, take 1 ul samples to carry out GC analyses.Carrier gas used is helium, and heat up journey Sequence is as follows：45 DEG C, 2 min；Then 100 DEG C are raised to 5 DEG C/min speed, keep 1 min；It is raised to again with 20 DEG C/min 220 DEG C, keep 2 min.Use CD-ACIDWAX（30 m×0.25 mm×0.25 um）Capillary column is divided compound From, and pass through Agilent Technologies 7890A（GC system）The 5975 MSD matter that chromatography of gases analyzer is matched somebody with somebody Spectrum detector is detected.
3rd, result
Bacillus subtilis engineering bacteriaB. subtilis 168-3 is as shown in Figure 5 to the tolerance of 1,2,3- trichloropropane.Withered grass Bacillus engineering bacteria culture supernatant crude enzyme liquid is as shown in Figure 6 to the degradation results of 1,2,3- trichloropropane.Bacillus subtilis Engineering Bureau bacterium is as shown in Figure 7 to the degradation of 1,2,3- trichloropropane, the trimethylewne chlorohydrin 3-s of 2,3- bis- and epoxychloropropane.
As a result show, after culture 25 days, engineering bacteria can be with 1 in degradable culture medium, 2,3- trichloropropanes, 2,3- Two trimethylewne chlorohydrin 3-s and epoxychloropropane（2mM）.Illustrate that the bacillus subtilis engineering bacteria built can degrade 1,2,3- trichlorines third Alkane and its intermediate product, realize the permineralization of 1,2,3- trichloropropanes.
SEQUENCE LISTING
<110>Zhongshan University
<120>The method of one kind bacillus subtilis engineering bacteria 1,2,3- trichloropropanes of degraded
<130>
<160> 35
<170> PatentIn version 3.3
<210> 1
<211> 882
<212> DNA
<213>The alkyl halide dehalogenation enzyme gene of synthesis（dhaA31）
<400> 1
atgtcagaaa ttggcacagg ctttccgttt gatccgcatt atgttgaagt tctgggcgaa 60
agaatgcatt atgttgatgt tggcccgaga gatggcacac cggttctgtt tctgcatggc 120
aatccgacat catcatatct gtggagaaat attattccgc atgttgcacc gtcacataga 180
tgcattgcac cggatctgat tggcatgggc aaatcagata aaccggatct ggattacttc 240
tttgatgatc atgttcgata cctggatgct ttcattgaag cactgggcct ggaagaagtt 300
gttctggtta ttcatgattg gggctcagca ctgggctttc attgggcaaa aagaaatccg 360
gaaagagtta aaggcattgc ttgtatggag tttattagac cgtttccgac atgggatgaa 420
tggccggaat ttgcaagaga aacatttcaa gcgtttagaa cagcagatgt tggcagagaa 480
ctgattattg atcaaaatgc ttttattgaa ggcgcactgc cgaaatatgt tgttagaccg 540
ctgacagaag ttgagatgga tcattataga gaaccgtttc tgaaaccggt tgatagagaa 600
ccgctgtgga gatttccgaa cgagctgccg attgcaggcg aaccggcaaa tattgttgca 660
ctggttgaag catatatgaa ttggctgcat caatcaccgg ttccgaaact gctgttttgg 720
ggcacaccgg gctttattat tccgccggca gaagcagcaa gactggcaga atcactgccg 780
aattgcaaaa cagttgatat tggccctggc ctgcattttc tgcaagaaga taatccggat 840
ctgattggct cagaaattgc aagatggctg ccggcactgt aa 882
<210> 2
<211> 293
<212> PRT
<213>Alkyl halide dehalogenase（haloalkanedehalogenase, DhaA31）
<400> 2
Met Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val
1 5 10 15
Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg
20 25 30
Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser
35 40 45
Tyr Leu Trp Arg Asn Ile Ile Pro His Val Ala Pro Ser His Arg
50 55 60
Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro
65 70 75
Asp Leu Asp Tyr Phe Phe Asp Asp His Val Arg Tyr Leu Asp Ala
80 85 90
Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His
95 100 105
Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro
110 115 120
Glu Arg Val Lys Gly Ile Ala Cys Met Glu Phe Ile Arg Pro Phe
125 130 135
Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln
140 145 150
Ala Phe Arg Thr Ala Asp Val Gly Arg Glu Leu Ile Ile Asp Gln
155 160 165
Asn Ala Phe Ile Glu Gly Ala Leu Pro Lys Tyr Val Val Arg Pro
170 175 180
Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Lys
185 190 195
Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro
200 205 210
Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Ala Tyr
215 220 225
Met Asn Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp
230 235 240
Gly Thr Pro Gly Phe Ile Ile Pro Pro Ala Glu Ala Ala Arg Leu
245 250 255
Ala Glu Ser Leu Pro Asn Cys Lys Thr Val Asp Ile Gly Pro Gly
260 265 270
Leu His Phe Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu
275 280 285
Ile Ala Arg Trp Leu Pro Ala Leu
290
<210> 3
<211> 765
<212> DNA
<213>The halide alcohol dehalogenase gene of synthesis（hheC）
<400> 3
atgtctacag caatcgttac aaatgttaaa cattttggcg gcatgggctc agcacttcgc 60
cttagcgaag caggacatac ggttgcttgt catgatgagt cttttaaaca aaaagatgaa 120
cttgaagcat ttgcagaaac gtatcctcaa cttaaaccta tgtcagaaca agaaccggca 180
gaactgatcg aagcagttac gtcagcctac ggacaagttg atgttctggt ttctaatgac 240
atctttgcac ctgaatttca accgatcgat aaatatgcag ttgaagatta tcgtggagca 300
gttgaagcac ttcaaattcg cccgtttgca ctggttaatg cagttgcatc tcagatgaaa 360
aaacgcaaat caggccatat tatctttatc acgagcgcaa caccgtttgg accgtggaaa 420
gaactgtcca cttatacgag cgcacgcgca ggcgcatcta cgctggcaaa tgcactgtct 480
aaagaattag gcgaatataa tattccggtt tttgcaatcg gacctaatta cttacatagc 540
gaagatagcc cttattttta tccgacggaa ccgtggaaaa cgaatcctga acatgttgca 600
catgttaaaa aagttacagc acttcaacgc ttaggcacac aaaaagaatt gggcgaactt 660
gttgcatttc tggcaagcgg ctcttgcgat tatctgacgg gccaagtatt ttggctggct 720
ggaggctttc ctatgatcga acgctggccg ggaatgccgg aataa 765
<210> 4
<211> 254
<212> PRT
<213>Halide alcohol dehalogenase（halohydrindehalogenase, HheC）
<400> 4
Met Ser Thr Ala Ile Val Thr Asn Val Lys His Phe Gly Gly Met
1 5 10 15
Gly Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys
20 25 30
His Asp Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala
35 40 45
Glu Thr Tyr Pro Gln Leu Lys Pro Met Ser Glu Gln Glu Pro Ala
50 55 60
Glu Leu Ile Glu Ala Val Thr Ser Ala Tyr Gly Gln Val Asp Val
65 70 75
Leu Val Ser Asn Asp Ile Phe Ala Pro Glu Phe Gln Pro Ile Asp
80 85 90
Lys Tyr Ala Val Glu Asp Tyr Arg Gly Ala Val Glu Ala Leu Gln
95 100 105
Ile Arg Pro Phe Ala Leu Val Asn Ala Val Ala Ser Gln Met Lys
110 115 120
Lys Arg Lys Ser Gly His Ile Ile Phe Ile Thr Ser Ala Thr Pro
125 130 135
Phe Gly Pro Trp Lys Glu Leu Ser Thr Tyr Thr Ser Ala Arg Ala
140 145 150
Gly Ala Ser Thr Leu Ala Asn Ala Leu Ser Lys Glu Leu Gly Glu
155 160 165
Tyr Asn Ile Pro Val Phe Ala Ile Gly Pro Asn Tyr Leu His Ser
170 175 180
Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu Pro Trp Lys Thr Asn
185 190 195
Pro Glu His Val Ala His Val Lys Lys Val Thr Ala Leu Gln Arg
200 205 210
Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala Phe Leu Ala
215 220 225
Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Trp Leu Ala
230 235 240
Gly Gly Phe Pro Met Ile Glu Arg Trp Pro Gly Met Pro Glu
245 250
<210> 5
<211> 885
<212> DNA
<213>The epoxide hydrolase of synthesis（EchA）Gene
<400> 5
atgacgattc gccgccctga agattttaaa cattatgaag ttcaacttcc tgatgttaaa 60
atccattatg ttcgcgaagg cgcaggcccg acgctgctgt tactgcatgg ctggccgggc 120
ttttggtggg aatggtctaa agttatcgga cctcttgcag aacattatga tgttatcgtt 180
ccggatctga gaggctttgg agattcagaa aaaccggatc ttaatgatct gtctaaatat 240
agccttgata aagcagcaga tgatcaagca gcacttctgg atgcactggg aatcgaaaaa 300
gcctatgttg ttggccatga ttttgcagca atcgttctgc ataaattcat ccgcaaatat 360
agcgatagag ttatcaaagc agcaatcttt gatcctatcc aaccggattt tggaccggtt 420
tattttggct taggacatgt tcatgaaagc tggtattcac aatttcatca actggatatg 480
gcagttgaag ttgttggctc tagccgcgaa gtttgcaaaa aatattttaa acatttcttt 540
gatcattgga gctatagaga tgaactgctt acagaagaag aacttgaagt tcatgttgat 600
aattgcatga aacctgataa tatccacggc ggctttaatt attatcgcgc aaatattcgc 660
cctgatgcag cactgtggac ggacctggat catacaatga gtgacctgcc ggttacgatg 720
atctggggac tgggcgatac atgcgttccg tatgcacctc ttatcgaatt tgttcctaaa 780
tattattcta attatacgat ggaaacgatc gaagattgcg gccattttct tatggttgaa 840
aaaccggaaa tcgcaatcga tagaatcaaa acggcattca gataa 885
<210> 6
<211> 294
<212> PRT
<213>Epoxide hydrolase（epoxide hydrolase, EchA）
<400> 6
Met Thr Ile Arg Arg Pro Glu Asp Phe Lys His Tyr Glu Val Gln
1 5 10 15
Leu Pro Asp Val Lys Ile His Tyr Val Arg Glu Gly Ala Gly Pro
20 25 30
Thr Leu Leu Leu Leu His Gly Trp Pro Gly Phe Trp Trp Glu Trp
35 40 45
Ser Lys Val Ile Gly Pro Leu Ala Glu His Tyr Asp Val Ile Val
50 55 60
Pro Asp Leu Arg Gly Phe Gly Asp Ser Glu Lys Pro Asp Leu Asn
65 70 75
Asp Leu Ser Lys Tyr Ser Leu Asp Lys Ala Ala Asp Asp Gln Ala
80 85 90
Ala Leu Leu Asp Ala Leu Gly Ile Glu Lys Ala Tyr Val Val Gly
95 100 105
His Asp Phe Ala Ala Ile Val Leu His Lys Phe Ile Arg Lys Tyr
110 115 120
Ser Asp Arg Val Ile Lys Ala Ala Ile Phe Asp Pro Ile Gln Pro
125 130 135
Asp Phe Gly Pro Val Tyr Phe Gly Leu Gly His Val His Glu Ser
140 145 150
Trp Tyr Ser Gln Phe His Gln Leu Asp Met Ala Val Glu Val Val
155 160 165
Gly Ser Ser Arg Glu Val Cys Lys Lys Tyr Phe Lys His Phe Phe
170 175 180
Asp His Trp Ser Tyr Arg Asp Glu Leu Leu Thr Glu Glu Glu Leu
185 190 195
Glu Val His Val Asp Asn Cys Met Lys Pro Asp Asn Ile His Gly
200 205 210
Gly Phe Asn Tyr Tyr Arg Ala Asn Ile Arg Pro Asp Ala Ala Leu
215 220 225
Trp Thr Asp Leu Asp His Thr Met Ser Asp Leu Pro Val Thr Met
230 235 240
Ile Trp Gly Leu Gly Asp Thr Cys Val Pro Tyr Ala Pro Leu Ile
245 250 255
Glu Phe Val Pro Lys Tyr Tyr Ser Asn Tyr Thr Met Glu Thr Ile
260 265 270
Glu Asp Cys Gly His Phe Leu Met Val Glu Lys Pro Glu Ile Ala
275 280 285
Ile Asp Arg Ile Lys Thr Ala Phe Arg
290
<210> 7
<211> 38
<212> DNA
<213>Primer Pgrac-fw
<400> 7
acactagcta gcagctattg taacataatc ggtacggg 38
<210> 8
<211> 29
<212> DNA
<213>Primer Pgrac-rv
<400> 8
taacgcggat ccttcctcct ttaattggg 29
<210> 9
<211> 28
<212> DNA
<213>Primer PhoD-fw
<400> 9
taacgcggat ccatggcata cgacagtc 28
<210> 10
<211> 30
<212> DNA
<213>Primer PhoD-rv
<400> 10
gcgccggtcg acagcattta cttcaaaggc 30
<210> 11
<211> 180
<212> DNA
<213>Pgrac promoter sequences
<400> 11
agctattgta acataatcgg tacgggggtg aaaaagctaa cggaaaaggg agcggaaaag 60
aatgatggat cactagaaaa ttttttaaaa aatctcttga cattggaagg gagatatgtt 120
attataagaa tagcggaatt gtgagcggat aacaattccc aattaaagga ggaaggatcc 180
<210> 12
<211> 168
<212> DNA
<213>PhoD signal peptides
<400> 12
atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 60
aacaatacgt ttgaccgccg caaatttatt caaggagcgg ggaagattgc aggactttct 120
cttggattaa cgattgccca gtcggttggg gcctttgaag taaatgct 168
<210> 13
<211> 348
<212> DNA
<213>DNA sequence dna component Pgrac-phoD cassette comprising promoter and signal peptide
<400> 13
agctattgta acataatcgg tacgggggtg aaaaagctaa cggaaaaggg agcggaaaag 60
aatgatggat cactagaaaa ttttttaaaa aatctcttga cattggaagg gagatatgtt 120
attataagaa tagcggaatt gtgagcggat aacaattccc aattaaagga ggaaggatcc 180
atggcatacg acagtcgttt tgatgaatgg gtacagaaac tgaaagagga aagctttcaa 240
aacaatacgt ttgaccgccg caaatttatt caaggagcgg ggaagattgc aggactttct 300
cttggattaa cgattgccca gtcggttggg gcctttgaag taaatgct 348
<210> 14
<211> 43
<212> DNA
<213>Primer dhaA31-fw
<400> 14
atccgcgtcg acatgtcaga aattggcaca ggctttccgt ttg 43
<210> 15
<211> 44
<212> DNA
<213>Primer dhaA31-rv
<400> 15
tattagcggc cgcttaatga tggtgatgat gatgcagtgc cggc 44
<210> 16
<211> 24
<212> DNA
<213>Primer MazE-fw
<400> 16
ttggcgcgcc cgataaccag aagc 24
<210> 17
<211> 29
<212> DNA
<213>Primer Amp-rv1
<400> 17
cgtggccagg cgcgccatga gtattcaac 29
<210> 18
<211> 27
<212> DNA
<213>Primer DR (up)-fw
<400> 18
ggactagtaa aattgaaaaa atggtgg 27
<210> 19
<211> 33
<212> DNA
<213>Primer DR (down)-rv
<400> 19
cgacgcgtga tccccctatg caagggttta ttg 33
<210> 20
<211> 29
<212> DNA
<213>Primer vpr (front)-fw
<400> 20
cgtggccagg atcattcgct ttctgcttg 29
<210> 21
<211> 30
<212> DNA
<213>Primer vpr (front)-rv
<400> 21
ggactagtat ccgactgtcc agccgttcgg 30
<210> 22
<211> 28
<212> DNA
<213>Primer vpr (back)-fw
<400> 22
cgacgcgtca ataacaaaga agttgagc 28
<210> 23
<211> 30
<212> DNA
<213>Primer vpr (back)-rv
<400> 23
ttggcgcgcc cggtcaaaac ctggcttgac 30
<210> 24
<211> 32
<212> DNA
<213>Primer hheC-fw
<400> 24
atccgcgtcg acatgtctac agcaatcgtt ac 32
<210> 25
<211> 38
<212> DNA
<213>Primer hheC-rv
<400> 25
taatagcggc cgcttaatga tgatgatgat gatgttcc 38
<210> 26
<211> 24
<212> DNA
<213>Primer MazE-fw
<400> 26
ttggcgcgcc cgataaccag aagc 24
<210> 27
<211> 29
<212> DNA
<213>Primer Amp-rv2
<400> 27
cgacgcgtgg cgcgccatga gtattcaac 29
<210> 28
<211> 29
<212> DNA
<213>Primer DR (up)-fw2
<400> 28
caggacgtcc aaaattgaaa aaatggtgg 29
<210> 29
<211> 33
<212> DNA
<213>Primer DR (down)-rv2
<400> 29
cgaggcctga tccccctatg caagggttta ttg 33
<210> 30
<211> 24
<212> DNA
<213>Primer epr (front)-fw
<400> 30
cgacgcgttg tatcagtcac tctg 24
<210> 31
<211> 32
<212> DNA
<213>Primer epr (front)-rv
<400> 31
caggacgtcc tggctccgat aatccctgcg ac 32
<210> 32
<211> 27
<212> DNA
<213>Primer epr (back)-fw
<400> 32
cgaggccttt caaagtcttc tccaagg 27
<210> 33
<211> 34
<212> DNA
<213>Primer epr (back)-rv
<400> 33
ttggcgcgcc cgtcttcaaa ttggtgctct tcac 34
<210> 34
<211> 43
<212> DNA
<213>Primer echA-fw
<400> 34
atccgcgtcg acatgacgat tcgccgccct gaagatttta aac 43
<210> 35
<211> 43
<212> DNA
<213>Primer echA-rv
<400> 35
ttatagcggc cgcttaatga tgatgatgat gatgtctgaa tgc 43

Claims (10)

1. a kind of alkyl halide dehalogenation enzyme gene, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.1.
2. a kind of alkyl halide dehalogenase, it is characterised in that amino acid sequence is as shown in SEQ ID NO.2.
3. a kind of halide alcohol dehalogenase gene, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.3.
4. a kind of halide alcohol dehalogenase, it is characterised in that amino acid sequence is as shown in SEQ ID NO.4.
5. a kind of epoxide hydrolase gene, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.5.
6. a kind of epoxide hydrolase, it is characterised in that amino acid sequence is as shown in SEQ ID NO.6.
7. the bacillus subtilis engineering bacteria of one kind 1,2,3- trichloropropanes of degraded, it is characterised in that build by the following method Arrive：First synthesis can degrade the alkyl halide dehalogenation enzyme gene of 1,2,3- trichloropropanes and its intermediate product, halide alcohol dehalogenase gene and Epoxide hydrolase gene, the method then knocked out by gene ectopic integration and resistant gene, said gene is integrated into In Bacillus subtilis genes group, acquisition can be with secreting, expressing alkyl halide dehalogenase, halide alcohol dehalogenase and epoxide hydrolase Bacillus subtilis engineering bacteria.
8. bacillus subtilis engineering bacteria according to claim 7, it is characterised in that its construction method includes following step Suddenly：
（1）Gene chemical synthesis：Synthesize alkyl halide dehalogenation enzyme genedhaA31st, halide alcohol dehalogenase genehheCWith epoxide hydrolase base CauseechA；
（2）Using Pgrac promoters and PhoD signal peptide sequences, build include respectivelydhaA31 genes,hheCGene andechA The bacillus subtilis integration vector of gene；
（3）By step（2）Integration vector conversion bacillus subtilis, pass through the side that gene ectopic integration and resistant gene are knocked out Method, willdhaA31 genes,hheCGene andechAGene is integrated into Bacillus subtilis genes group successively, and structure obtains dividing Secrete the bacillus subtilis engineering bacteria of expression alkyl halide dehalogenase, halide alcohol dehalogenase and epoxide hydrolase.
9. the bacillus subtilis engineering bacteria described in claim 7 or 8 is in 1,2,3- trichloropropanes, the chloro- 1- third of 2,3- bis- of degrading Application in terms of alcohol and/or epoxychloropropane, or bacillus subtilis engineering bacteria described in claim 7 or 8 are preparing 1,2, Application in terms of the degraded preparation of 3- trichloropropanes, the trimethylewne chlorohydrin 3-s of 2,3- bis- and/or epoxychloropropane.
The method of 1,2,3- trichloropropanes 10. one kind is degraded with bacillus subtilis engineering bacteria, it is characterised in that will using right Seek bacillus subtilis engineering bacteria 1,2, the 3- trichloropropanes of degraded described in 7 or 8.
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CN111094578A (en) * 2017-09-28 2020-05-01 拜耳股份公司 Preparation method of chiral α halogenated alkanoic acid
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