CN106811427B - Angle dioxygenase gene dpeA1A2 and its application - Google Patents
Angle dioxygenase gene dpeA1A2 and its application Download PDFInfo
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Abstract
The invention discloses angle dioxygenase gene dpeA1A2 and its applications.The angle dioxygenase small subunit encoding gene dpeA2 and catenation sequence GATG between the two that size shown in a kind of angle dioxygenase gene dpeA1A2, angle dioxygenase large subunit encoding gene dpeA1, the SEQ ID NO.2 that the size shown in SEQ ID NO.1 is 1383bp is 561bp are formed.DpeA1A2BC holoenzyme provided by the invention can in 72hr degradable 100mg/L diphenyl ether；Furthermore DpeA1A2BC holoenzyme can also in 72hr degradable 100mg/L 2- phenoxy benzoic acid (2-PBA).Therefore angle dioxygenase gene dpeA1A2 is applied in building degradation diphenyl ether or 2- phenoxy benzoic acid genetically modified crops.DpeA1A2BC holoenzyme is applied in degradation diphenyl ether and 2-PBA.
Description
Technical field
The invention belongs to application environment microorganism and agriculture fields, are related to angle dioxygenase gene dpeA1A2 and its answer
With.
Background technique
Chemical pesticide is to ensure one of the important means of China's agricultural good harvest, there is very important work in agricultural production
With.Pesticide refers to the chemistry examination of the harmful organisms such as disease pest, the weeds for being applied to that prevention and treatment harms the crops and coordinate plant growth
The general name of agent.But due to a large amount of uses of severe toxicity, high residue, pesticide difficult to degrade for a long time, we, which are faced with, is continuously increased
Soil, underground water and atmosphere pollution by pesticides environmental problem.And many pesticides are synthesized by intermediate, so degradation agriculture
Medicine intermediate has eurytopicity and high efficiency.Pollution by pesticides degradation technique can be divided into thermal degradation, light degradation, chemical degradation and biology
Degradation.Biodegrade is exactly pesticide to be decomposed into the non-toxic or low-toxic compound of small molecule by the effect of biology, and finally degrade
For water.Wherein microorganism remediation technology has the characteristics that high-efficient, effect is good, more green, is the preferably square of pesticide recovery technique
Method.
The intermediate of diphenyl ether and 2- phenoxy benzoic acid (2-PBA) and common pesticide herbicide, also being important
Learn compound probability intermediate.Its derivative and halogen substituents can synthesize the chemical industry such as herbicide, agrochemical pesticide and drug
Product.It obtains diphenyl ether and 2-PBA degradation bacteria strains and degrading genes and is administering herbicide residue, eliminate in its phytotoxicity technical research
It has the effect that and degrading genes is imported crop by modern biotechnology and construct corresponding herbicide by function, (one)
Resistant transgenic crop, (two) by Modern microbiological fermentation technique by diphenyl ether and 2-PBA pesticide intermediate degradation bacteria strains and
Degradation bacterial agent is made in gene or enzyme preparation can be by diphenyl ether in soil and 2-PBA residue degrading.In addition, diphenyl ether and 2-PBA
Degrading genes can also be used in the bioconversion of chemical dyestuff intermediate and pharmaceutical synthesis and the life of diphenyl ether compound pollution
Object reparation.Therefore diphenyl ether and 2-PBA degrading genes eliminate such herbicide intermediate phytotoxicity, environmental pollution it is biological prosthetic and
There is very important theoretical and application value in field of bioconversion.
The double oxygenations (Angular dioxygenase) of angle, which are that a kind of distinctive degradation of microorganism is certain, has angled arrangement
Aromatic hydrocarbons pollutant (such as dioxin, carbazole, fluorenes, dibenzothiophenes, diphenyl ether and dibenzofurans compound) mode.
Have on the phenyl ring of these compounds and be connected to O, N atom (fluorenes connect with dibenzothiophenes be C) on a C atom, forms one
A angle, therefore this C atom is angle C atom.
The angle dioxygenase of catalysis diphenyl ether compound 3- phenoxy benzoic acid (3-PBA) ether bond rupture is reported altogether at present
Road two.One is the PobAB in Pseudomonas pseudoalcaligenes POB310, and it is non-to belong to I- type Rieske
Heme iron oxidizing ferment (Rieske non-heme iron oxygenase, RHO) (Kweon et al., 2008), there is 2 groups
Part: a terminal oxidase and a FNRCType reductase (Dehmel et al., 1995), PobAB attack diphenyl ether
1,6 carbon atoms for closing the benzoic acid part of object 3-PBA cause ether bond rupture to generate phenol and protocatechuic acid (see Fig. 1).It is another
A is the PbaA1A2BC found from Sphingobium wenxiniae JZ-1, belongs to IV- type RHOs, there is 3 components: one
A terminal oxidase, [2Fe-2S]-type ferredoxin and GR- type reductase (Wang et al., 2014),
PbaA1A2BC attacks 1 ', 2 ' position carbon atoms of the benzene part of 3-PBA, causes the ether bond rupture of 3- phenoxy benzoic acid to generate adjacent
Benzenediol and 3- hydroxybenzoic acid (see Fig. 2).
The side dioxygenase gene cluster of diphenyl ether compound of capable of degrading also has been reported that.Frank Prefer in 1989
Deng the double oxygenation approach in side that (Arch Microbioal) reports diphenyl ether in Pseudomonas bacterial strain, they pass through mass spectrum
And liquid phase detects intermediate product 2,3- dihydroxy diphenyl ether, the substance then hydrolyze as phenol and hexa-atomic keto carboxylic acid (see
Fig. 3).Hexa-atomic keto carboxylic acid is not continued degradation.They have found that the 2,3- dihydroxybiphenyl in Pseudomonas ET4 bacterium is double simultaneously
The ring-opening reaction of oxygenation enzymatic 2,3- dihydroxy diphenyl ether.
At present both at home and abroad it has been reported that the gene or gene cluster of three degradation diphenyl ether compounds, however can degrade
There is not been reported for the microorganism of 2-PBA.Present invention firstly discovers that the sheath ester bacterium SC-3 of one plant of 2-PBA that can degrade, and pass through
Research is cloned into the gene cluster of degradation 2-PBA for the first time, has filled up the blank in this domestic and international field.Degradation diphenyl ether in the present invention
Effect gene and reported gene similarity are low, product and reported difference after degradation, are a kind of novel degradations two
The gene of phenylate.So sheath ester bacterium SC-3 and its angle dioxygenase gene dpeA1A2 are novel economic benefits and social benefits pesticide intermediates
Degradation bacteria strains and degrading genes.
Summary of the invention
The object of the present invention is to provide the eggs of the angle dioxygenase gene dpeA1A2 and its coding of diphenyl ether and 2-PBA
White matter and its application, the angle dioxygenase gene can be used for constructing the genetically modified crops of degradation diphenyl ether and 2-PBA, it is also possible to
The bioconversion of herbicide removal and chemical dyestuff and pharmaceutical synthesis in soil, water body.
It is a further object of the present invention to provide the applications of the gene.
The purpose of the present invention is achieved through the following technical solutions:
The degradation bacteria SC_3 of one plant of diphenyl ether and 2-PBA is preserved in China typical culture collection on June 30th, 2015
Center, deposit number are as follows: CCTCC NO:M2015417.
By dilution plate rubbing method, separation screening is dropped to one plant of 2-PBA from the pregnant solution with 2-PBA degradation effect
Bacterial strain is solved, SC_3 is named as.This bacterial strain is bacterial strain used in this patent.Bacterial strain SC_3 can in 3 days degradable 100mg
L-12-PBA and diphenyl ether.The cell transmission electron microscope photo of bacterial strain SC_3 is as shown in Figure 5.Bacterial strain SC_3 R2A, LB,
30 DEG C of well-growns on NA and TSA plate, bacterium colony are in yellow, the smooth of the edge, bacterium colony projection, Gram-negative, rod-short
Cell, it is aerobic, there is polar flagella.
The 16S rRNA gene order of SC_3 and Sphingobium category Characterization are nearest, wherein with Sphingobium
abikonense KCTC 2864TSimilitude is up to 97%.Bacterial strain SC_3 and the closer strain of affinity are made into adjacent method
Phylogenetic tree (Fig. 6), bacterial strain SC_3 is together with Sphingobiun category type strain cluster on phylogenetic tree, but shape
At an individual branch.Therefore, it is developed in conjunction with colonial morphology characteristic, physiological and biochemical property and 16S rRNA genic system
Tree analysis, bacterial strain SC_3 be accredited as Sphingobium belong to (Sphingobium phenoxybenzoativorans).Bacterial strain is protected
China typical culture collection center is ensconced, deposit number is CCTCC NO:M2015417, and the deposit date is June 30 in 2015
Day.
Analysis diphenyl ether and 2-PBA (Fig. 7) the result shows that the crude enzyme liquid of bacterial strain SC-3 can degrade.
The strategy that the gene of the angle dioxygenase of clone's degradation diphenyl ether and 2-PBA is taken is genome sequencing, is led to
It crosses and genomic information analysis and dioxygenase gene relevant to the degradation of reported aromatic compound is compared to clone target
Gene.The dioxygenase DpeA1A2 of catalysis diphenyl ether compound degradation of the invention should be with catalysis class on amino acid sequence
Have certain similitude like the reported dioxygenase of reaction, thus it is quasi- by use it has been reported that dioxygenase amino acid sequence
Column and bacterial strain SC_3 genome alignment, discovery have the gene of certain similitude with these dioxygenase amino acid sequences.It utilizes
At present it has been reported that 10 dioxygenase amino acid sequences and bacterial strain SC_3 genome alignment.As a result, it has been found that bacterial strain SC_3 gene
Group has 3 positions and has reported that angle dioxygenase has similitude, further analyzes the DNA sequence dna of this 3 positions, will include
The sequence (respectively staying 2-3kb before and after gene) of possible dioxygenase gene is cloned into wide host cell pBBR1MCS-5 and imports mutation
Bacterial strain and without in the bacterial strain Sphingobium wenxinae JZ-1 of diphenyl ether degradation capability, whether measurement transformant obtains
It degrades diphenyl ether ability (see Fig. 5).
The transformant of can degrade diphenyl ether and 2-PBA is obtained with strategy above, is finally determined that coding angle is double and is added
The unnamed gene of oxygenase is dpeA1A2.This is the gene for being cloned into can degrade diphenyl ether and 2-PBA for the first time.The gene is by SEQ
Size shown in ID NO.1 is shown in angle dioxygenase large subunit encoding gene dpeA1, the SEQ ID NO.2 of 1383bp
The angle dioxygenase small subunit encoding gene dpeA2 and catenation sequence GATG composition between the two that size is 561bp.
The dioxygenase DpeA1A2 for the catalysis diphenyl ether compound open loop that the present invention reports is by DpeA1 and DpeA2 two
One angle dioxygenase of subunit composition, gene and enzyme amino acid sequence and catalyst mechanism are different from three kinds of above-mentioned report
It is catalyzed the dioxygenase of diphenyl ether degradation.DpeA1 and reported diphenyl ether dioxygenase large subunit PobA, PbaA1 and BphA1
Homology on amino acid levels only has 37%, 36%, 37% respectively.DpeA2 and reported diphenyl ether dioxygenase are small
The homology of subunit PobB, PbaA2 and BphA2 on amino acid levels only has 33%, 35%, 40% respectively.In catalyst mechanism
On, DpeA1A2 causes phenyl ring to crack in 1,2 of the one of phenyl ring of diphenyl ether double oxygenations, generate product 6- oxo -2,4- oneself
Dienoic acid phenol ester (see Fig. 4).
The angle dioxygenase protein D peA1A2 of angle dioxygenase gene dpeA1A2 coding of the present invention, by ammonia
Base acid sequence are as follows: angle shown in angle dioxygenase large subunit DpeA1 and SEQ ID NO.4 shown in SEQ ID NO.3 is double
Oxygenase small sub-unit DpeA2 composition.
DpeA1A2BC holoenzyme angle dioxygenase large subunit DpeA1, SEQ ID NO.4 institute as shown in SEQ ID NO.3
Ferredoxin DpeB and SEQ ID NO.14 shown in angle dioxygenase small subunit DpeA2, the SEQ ID NO.10 shown
Shown in reductase DpeC composition.
The encoding gene of the DpeA1A2BC holoenzyme, the angle dioxygenase large subunit as shown in SEQ ID NO.1 are compiled
Shown in angle dioxygenase small subunit encoding gene dpeA2, SEQ ID NO.9 shown in code gene dpeA1, SEQ ID NO.2
Ferredoxin encoding gene dpeB and SEQ ID NO.13 shown in reductase dpeC composition.
Contain the angle dioxygenase large subunit encoding gene dpeA1 or angle dioxygenase small subunit encoding gene
The recombinant expression carrier of dpeA2.
Containing the angle dioxygenase large subunit encoding gene dpeA1 or angle dioxygenase small subunit encoding gene
The genetic engineering bacterium of dpeA2.
Application of the degradation bacteria SC_3 of the present invention in degradation diphenyl ether and 2-PBA.
Application of the DpeA1A2BC holoenzyme in degradation diphenyl ether and 2-PBA.
Application of the DpeA1A2BC holoenzyme in removal soil, water body in diphenyl ether and 2-PBA.
Application of the encoding gene of the DpeA1A2BC holoenzyme in degradation diphenyl ether and 2-PBA.
Beneficial effects of the present invention are as follows:
1. genome sequencing of the invention and gene comparison method successfully from bacterial strain SC-3 (CCTCC NO:
M2015417 angle dioxygenase enzyme gene dpeA1A2 is cloned in).Show that the gene is one in GenBank comparison result
New gene, overall length (from initiation codon to terminator codon) are 1948, encode 647 amino acid.
2. DpeA1A2BC holoenzyme provided by the invention can in 72hr degradable 100mgL-1Diphenyl ether and 2-
PBA, dpeA1A2 can be used for constructing degradation diphenyl ether and 2-PBA genetically modified crops, it can also be used to remove soil, weeding in water body
The bioconversion of agent and pharmaceutical synthesis has very important theoretical and application value.
Detailed description of the invention
The schematic diagram of Fig. 1 PobAB catalysis diphenyl ether compound 3- phenoxy benzoic acid ether bond rupture
The schematic diagram of Fig. 2 PbaA1A2BC catalysis diphenyl ether compound 3- phenoxy benzoic acid ether bond rupture
The side Fig. 3 dioxygenase gene BphAaAbAcAd is catalyzed diphenyl ether degradation schematic diagram
Fig. 4 angle dioxygenase gene dpeA1A2 is catalyzed diphenyl ether degradation schematic diagram
The policy map of Fig. 5 angle dioxygenase gene dpeA1A2 clone
The SDS-PAGE electrophoresis of DpeA1, DpeA2, DpeB and the DpeC of Fig. 6 after purification are composed
The LC/MS map of Fig. 7 DpeA1A2BC holoenzyme catalytic degradation diphenyl ether
A: the LC-MS map of diphenyl ether
The LC-MS map of the catabolite phenol of B:DpeA1A2BC holoenzyme catalytic degradation diphenyl ether
The LC- of the catabolite 6- oxo -2,4- hexadienoic acid phenol ester of C:DpeA1A2BC holoenzyme catalytic degradation diphenyl ether
MS map
The LC/MS map of Fig. 8 DpeA1A2BC holoenzyme catalytic degradation 2-PBA
The LC-MS map of A:2-PBA
The LC- of the catabolite 6- oxo -2,4- hexadienoic acid phenol ester of B:DpeA1A2BC holoenzyme catalytic degradation 2-PBA
MS map
The LC-MS map of the catabolite phenol of C:DpeA1A2BC holoenzyme catalytic degradation diphenyl ether
The biochemical reaction approach of Fig. 9 DpeA1A2BC holoenzyme degradation 2-PBA.
Biomaterial preservation information
Sheath ester bacterium SC-3 (Sphingobium phenoxybenzoativorans.sp.nov.SC_3), is stored in China
Type Tissue Collection, address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:M2015417, preservation day
Phase is on June 30th, 2015.
Specific embodiment
The clone of 1. angle dioxygenase gene dpeA1A2 of embodiment
The extraction of 1.1 bacterial genomes total DNAs
After SC-3 (CCTCC NO:M2015417) mass propgation, extract the SC-3's of high-purity, large fragment using CTAB method
Genome DNA is dissolved in TE buffer (pH8.0), is placed in -20 DEG C of preservations, specific method is with reference to volumes such as F Ao Sibai
" fine works molecular biology experiment guide ".
The building of 1.2 recombinant plasmids
Expand the segment comprising doubtful 2-PBA (diphenyl ether) degrading genes cluster respectively by PCR, enzyme is connected to broad host range plasmid
On pBBR1MCS-5, construction recombination plasmid.By genome analysis and sequence alignment, three suspected target dioxygenases are filtered out
Gene carries out functional verification experiment, finally finds that one of gene is responsible for the degradation of 2-PBA and diphenyl ether.
Using SC_3 genomic DNA as template, design primer is used to expand doubtful dioxygenase gene segment.The primer
It is as follows:
Used primer is tested in 1 functional verification of table
PCR amplification system: 2 × GC Buffer, 25 μ L, dNTPs (2.5mmolL-1) 8 μ L, primer (25pmol μ L-1) each 1 μ L, SC_3 total DNA (about 50ng μ L-1) 2 μ L, LA-Taq archaeal dna polymerase (5U μ L-1) 0.5 μ L, add H2O to 50 μ
L。
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 1kb min-1, 30 circulations；72℃5min.
With Xba I and Hind III double digestion PCR product and pBBR1MCS-5 plasmid.Endonuclease reaction system is as follows: PCR is produced
Each 5 μ L of 2.5 μ L, 10 × M buffer of 40 μ L, Xba I and Hind III of object/plasmid.10 μ L 10 are added after 37 DEG C of overnight digestions
× Loading buffer terminates reaction.Use the DNA fragmentation and plasmid of TAKARA plastic recovery kit recycling double digestion；Then
16 DEG C of enzymes of recycling plasmid and DNA fragmentation are even stayed overnight, constructing function verifies plasmid pBBR1, pBBR2 and pBBR3；Enzyme-linked product turns
Change into competence E.coli DH5 α；Gained converts Strain Designation DH5 α (pBBR1), DH5 α (pBBR2) and DH5 α (pBBR3).
The building of 1.3 recombinant bacterial strains
By donor bacterium DH (pBBR1), DH (pBBR2) and DH (pBBR3), recipient bacterium Sphingobium wenxiniae
JZ-1 and Sphingomonas sp.RW1, auxiliary bacterium E.coli HB101 (PRK600) are respectively connected to containing corresponding antibiotic
In LB liquid medium, culture to logarithmic phase is resuspended thallus with sterile water after centrifugation and presses donor bacterium: recipient bacterium: auxiliary bacterium=1:
The volume ratio of 2:1 is uniformly mixed in centrifuge tube.Sterilised membrane filter is placed on the LB plate of non-resistant, Mixed Microbes are then drawn
200 μ L of liquid is added on filter membrane, and 30 DEG C are washed the thallus on lower filter membrane after stationary culture 4 days, with liquid LB, and concussion mixes, and takes 100 μ L
Bacterium solution is coated on selective resistance LB plate (Str and Gm).Cultivate 4 days pickings be transferred to plasmid joint element it is dual anti-in Gm and Sm
Flat lining out purifying is in case subsequent degradation function verification test.
1.4 different expression bacterial strains verify 2-PBA and diphenyl ether degradation function
By DH5 α (pBBR1/pBBR3/pBBR5), Sphingobium wenxie JZ-1 (pBBR1/pBBR3/pBBR5)
It is seeded to respectively from picking single colonie on LB plate with Sphingomonas sp.RW1 (pBBR1/pBBR3/pBBR5) corresponding double
In the LB liquid medium of the 100mL of resistance, in the shaking table of 150rpm after culture to logarithmic growth phase, it is centrifuged and uses sterile water
100mgL is respectively connected to after thallus is resuspended-12-PBA and diphenyl ether minimal medium in, DH5 α is in 37 DEG C, other bacterial strains
It is cultivated in 30 DEG C.2-PBA sample directly uses 0.45 μm of membrane filtration after culture 72h, and diphenyl ether sample need to use the bodies such as chloroform
0.22 μm of filter membrane is crossed after product extraction.The sample of preparation is used for the detection of HPLC and LC-MS.Liquid phase detection discovery, imports recombination matter
The recombinant bacterium Sphingobium wenxie JZ-1 (pBBR3) and Sphingomonas sp.RW1 (pBBR3) of grain pBBR3 can
Enough degradation 2-PBA and diphenyl ether, and phenol product and 6- oxo -2,4- hexadienoic acid phenol ester are also generated in degradation process
(Fig. 4-8)；2-PBA and diphenyl ether and the recombinant bacterial strain for being transferred to pBBR1 and pBBR5 cannot all degrade.The result shows that recombination imports
Third section dioxygenase gene cluster so that this kind of bacterial strain is obtained the degradation function of 2-PBA, diphenyl ether, therefore primarily determine the base
Because cluster should be exactly the gene for encoding 2-PBA and diphenyl ether angle dioxygenase, it is named as dpeA1A2.But import weight
The E. coli recombinant stain E.coli DH5 α (D3) of group plasmid pBBR3 cannot but degrade 2-PBA and diphenyl ether), thus it is speculated that having can
It can be because lacking suitable electronic transfer chain component i.e. iron oxygen required for this dioxygenase catalytic action also in Escherichia coli
Albumen and ferredoxin reductase, thus cannot be for caused by the terminal oxidase transfer components electronics of this dioxygenase.
High efficient expression of the 2. angle dioxygenase gene dpeA1A2 of embodiment in BL21 (pET-29a (+))
E. coli recombinant stain E.coli DH5 α (dpeA1A2) due to importing dpeA1A2 gene cannot degrade 2-
PBA and diphenyl ether, we suspect suitable electronic transfer chain required for lacking this dioxygenase catalytic action in Escherichia coli
Component, that is, ferredoxin and ferredoxin reductase lead to its purpose substrate that cannot degrade.In addition, gene comparison does not also exist
DpeA1A2 surrounding genes find relevant electron transmission chain gene.Therefore, we find associated electrical by gene alignments
Transmitting chain gene simultaneously verifies its function in the middle high efficient expression that carries out of BL21 (pET-29a (+)) together with dpeA1A2.
The amplification of 2.1 dpeA1A2 and electron transmission chain gene
Using bacterial strain SC_3 genome as template, dpeA1A2 and possible is expanded by high fidelity enzyme with corresponding primer respectively
Ferredoxin dpeB and reductase gene dpeC sequence.
The forward primer of dpeA1 gene is expanded as shown in SEQ ID NO.5, reverse primer is as shown in SEQ ID NO.6.
The forward primer of dpeA2 gene is expanded as shown in SEQ ID NO.7, reverse primer is as shown in SEQ ID NO.8.
The forward primer of ferredoxin encoding gene dpeB is expanded as shown in SEQ ID NO.11, reverse primer such as SEQ
Shown in ID NO.12.
The forward primer of amplification reduction enzyme coding gene dpeC is as shown in SEQ ID NO.15, reverse primer such as SEQ ID
Shown in NO.16.
PCR system:
5×Prime STAR HSBuffer(Mg2+)10.0μL
dNTP(2.5mmol·L-1)4.0μL
Forward primer (25pmol μ L-1)1.0μL
Reverse primer (25pmol μ L-1)1.0μL
PrimeSTAR DNA Polymerase(2.5U·μL-1)0.5μL
Template DNA (about 50ng μ L-1)2.0μL
ddH2O 31.5μL
50 μ L of total system
PCR reaction condition:
The building of 2.2 expression vectors
Nde I and Hind III double digestion dpeA2 gene, dpeA1, dpeB and dpeC base are used after PCR product purification and recovery
Because carrying out double digestion with Nde I and Xho I.Digestion system (50 μ L) is as follows:
PCR product: 30 μ L
Nde I:2.5μL
Hind III:2.5μL
10×HBuffer:5μL
ddH2O:10μL
After 37 DEG C of digestion 8h, the corresponding DNA of gel extraction is even stayed overnight with 16 DEG C of enzymes of pET29a plasmid of same double digestion.Enzyme
Disjunctor system (10 μ L) is as follows:
pET29a:1.5μL
DNA fragmentation: 7 μ L
T4 ligase: 0.5 μ L
Buffer:1 μ L
Enzyme-linked product is then converted into BL21 (DE3) competent cell, is coated on 50mgL-1On the LB plate of Km resistance,
37 DEG C of culture to single colonies are grown.After picking single colonie LB test tube spreads cultivation, whether PCR verifying segment is inserted into and sequence verification segment
It is whether correct.The recombinant vector bacterium of building is respectively designated as BL29DpeA1, BL29DpeA2, BL29DpeB and BL29DpeC.
2.3 the expression and purity of recombinant bacterium
Recombinant bacterial strain BL29DpeA1, BL29DpeA2, BL29DpeB and BL29DpeC are respectively at 50mgL-1Km resistance
The flat lining out purifying of LB, subsequent picking single colonie are seeded to 50mgL-137 DEG C in the 5ml LB test tube of Km, 200rpm culture
12h, is seeded to the LB liquid medium of 400mL by 1% inoculum concentration, and 37 DEG C, 200rpm cultivates to OD600After 0.6,0.5mM is added
FeCl3With 16 DEG C of low temperature induction 12h of IPTG.4 DEG C, 12,000g pelleted by centrifugation 10min collect thallus, then use 20mL PBS
(pH 7.0) thallus is resuspended in buffer, on ice ultrasonic disruption thalline 30min.Then in 4 DEG C, 12,000g centrifugation 20min are collected
Supernatant.Supernatant is protein induced rear for further purifying by SDS-PAGE detection confirmation.The above process is from ultrasonic disruption
It rises and operates at low temperature.Purifying protein electrophorogram is shown in Fig. 6.
2.4 DpeA1A2BC holoenzymes measure the degrading activity of 2-PBA and diphenyl ether
Using the system of determination angle dioxygenase holoenzyme DpeA1A2BC of 1mL to the degrading activity of 2-PBA and diphenyl ether,
Specific system is as follows: 0.5mM 2-PBA (or diphenyl ether), 2 μ g DpeA1,2 μ g DpeA2,1.8 μ g DpeB, 4.2 μ g DpeC,
1mM NADH+、0.5mM Fe2+With 1mM Mg2+, buffer system is 20mM PBS (pH7.0)；Three control groups are set simultaneously: right
According to a group 1:0.5mM 2-PBA (or diphenyl ether), 1.8 μ g DpeB, 1mM NADH+、0.5mM Fe2+、1mM Mg2+, buffer system is
20mM PBS(pH7.0)；Control group 2:0.5mM 2-PBA (or diphenyl ether), 4.2 μ g DpeC, 1mM NADH+、0.5mM Fe2+、
1mM Mg2+, buffer system is 20mM PBS (pH7.0)；Control group 3:0.5mM 2-PBA (or diphenyl ether), 1.8 μ g DpeB,
4.2μg DpeC、1mM NADH+、0.5mM Fe2+With 1mM Mg2+, buffer system is 20mM PBS (pH7.0).It is anti-by what is added
It answers system to be placed in 30 DEG C of water-baths and reacts 2h, then boiling water 3min terminates reaction.The bodies such as addition after sample is freeze-dried
Long-pending methanol sample dissolution, HPLC detection substrate concentration, which changes and passes through LC-MS, identifies catabolite.Enzyme activity unit definition
Are as follows: the amount that enzyme required for 1nmol substrate is consumed in one minute is indicated with U.It is found by liquid phase measurement and Mass Spectrometric Identification, only
The group of addition DpeA1A2BC holoenzyme could degrade 2-PBA and diphenyl ether, and catabolite liquid phase and mass spectrogram are shown in Fig. 7 and Fig. 8.
Cannot degrade 2-PBA and diphenyl ether under the conditions of DpeB and DpeC one pack system and DpeB and DpeC two-component.
Claims (8)
1. a kind of angle dioxygenase gene dpeA1A2, it is characterised in that: the angle dioxygenase as shown in SEQ ID NO.1
Angle dioxygenase small subunit encoding gene dpeA2 and the two shown in large subunit encoding gene dpeA1, SEQ ID NO.2
Between catenation sequence GATG composition.
2. the angle dioxygenase protein D peA1A2 of angle dioxygenase gene dpeA1A2 coding described in claim 1,
It is characterized in that: being angle dioxygenase large subunit DpeA1 and SEQ ID NO.4 shown in SEQ ID NO.3 as amino acid sequence
Shown in angle dioxygenase small subunit DpeA2 composition.
3.DpeA1A2BC holoenzyme, angle dioxygenase large subunit DpeA1, the SEQ ID NO.4 institute as shown in SEQ ID NO.3
Ferredoxin DpeB and SEQ ID NO.14 shown in angle dioxygenase small subunit DpeA2, the SEQ ID NO.10 shown
Shown in reductase DpeC composition.
4. the encoding gene of DpeA1A2BC holoenzyme as claimed in claim 3, it is characterised in that: the angle as shown in SEQ ID NO.1
Spend angle dioxygenase small subunit encoding gene shown in dioxygenase large subunit encoding gene dpeA1, SEQ ID NO.2
The coding of reductase shown in ferredoxin encoding gene dpeB and SEQ ID NO.13 shown in dpeA2, SEQ ID NO.9
Gene dpeC composition.
5. containing shown in the angle dioxygenase large subunit encoding gene dpeA1 or SEQ ID NO.2 as shown in SEQ ID NO.1
Angle dioxygenase small subunit encoding gene dpeA2 recombinant expression carrier.
6. containing shown in angle dioxygenase large subunit encoding gene dpeA1 or SEQ ID NO.2 shown in SEQ ID NO.1
The genetic engineering bacterium of angle dioxygenase small subunit encoding gene dpeA2.
7. application of the DpeA1A2BC holoenzyme as claimed in claim 3 in degradation diphenyl ether and 2- phenoxy benzoic acid.
8. the encoding gene of DpeA1A2BC holoenzyme as claimed in claim 4 is in degradation diphenyl ether and 2- phenoxy benzoic acid
Using.
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| PCT/CN2016/102881 WO2017092513A1 (en) | 2015-11-30 | 2016-10-21 | Angular dioxygenase gene dpea1a2 and use thereof |
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| Title |
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| Sphingobium phenoxybenzoativorans sp. nov.， a 2-phenoxybenzoic-acid-degrading bacterium;Shu Cai等;《International Journal of Systematic and Evolutionary Microbiology》;20150630;第65卷(第6期);第1986-1991页，参见摘要，第1986页右栏第2段-第1987页 * |
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