CN116426547A - Artificial transformation 2,4-dichlorophenol hydroxylase gene and application thereof - Google Patents

Abstract

The invention relates to an artificially modified 2,4-dichlorophenol hydroxylase gene and application, wherein MIX-tfdB and expressed protein thereof, CPs clearance experiments show that MIX-TfdB has higher enzyme activity on 2,4-DCP, 2,5-DCP and 3,4,5-TCP and higher removal rate; 3,4-DCP and 2,3,4-TCP have high removal rate although the enzyme activity is not high; in addition, the CTX3-tfdB gene and the expression protein thereof also show higher CPs removal rate.

Description

Artificial transformation 2,4-dichlorophenol hydroxylase gene and application thereof

Technical Field

The invention belongs to the field of genes, and particularly relates to an artificially modified 2,4-dichlorophenol hydroxylase gene and application thereof.

Background

With the development of industry, chlorophenols are widely used as pesticides, herbicides and preservatives, and have strong bioaccumulation toxicity and three-cause effect. Chlorophenols (CPs) pollute the environment, have large toxicity, and particularly have the property of being difficult to degrade, which always becomes the difficulty of environmental control, and are listed in a blacklist for preferentially controlling pollutants. CPs are mostly aromatic compounds having a cyclic structure, and hydroxylation, one of the most critical steps in the degradation of aromatic compounds, is catalyzed by hydroxylases. Bioremediation is a hotspot for degradation of chlorophenols pollutants, and research on the biodegradation pathway of the herbicide 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the environment has received attention. Thus, many 2,4-D degrading bacteria were isolated from different environments, 2,4-dichlorophenol hydroxylase was originally discovered from 2,4-D degrading bacteria Cupriavidus necator JMP 134. As the 2,4-D degradation mechanism of the bacterium is studied intensively, the enzyme is found to be located on a plasmid contained in C.necator JMP134, and the coding gene is named as tfdB. 2,4-dichlorophenol hydroxylase (2, 4-dichlorophenol hydroxylase, also known as 2,4-dichlorophenol 6-monooxygenase, tfdB) is a flavin hydroxylase, non-covalently bound to a FAD molecule.

TfdB is an enzyme involved in bacterial degradation of 2,4-D, a key step performed by this enzyme is the initial hydroxylation of the benzene ring, and 2,4-DCP (2, 4-dichlorophenol) is considered a natural substrate for TfdB. The activity in all the currently reported substrates is relatively high and the specificity is good. The hydroxylase acts by adding one oxygen atom of molecular oxygen to the aromatic ring and the other oxygen atom participating in the reduction to water, while this step is a conversion of 2,4-DCP to 3, 5-dichloropcatechol depending on the coenzyme NADPH as electron donor, and then the resulting product is finally put into the TCA cycle under the action of a series of hydroxylases and reductases. Only after one or more hydroxyl groups are introduced on the aromatic ring under the action of hydroxylase, the compound is favorable for ring opening and decomposition, and TfdB has certain degradation activity on chlorophenols and homologs thereof. Currently, most of the hydroxylases reported in research do not have the characteristic of substrate universality and are not suitable for industrial treatment of pollutants. Therefore, the construction of the efficient recombinant TfdB has important significance for degrading chlorophenol pollutants by microorganisms.

Disclosure of Invention

The invention provides a2, 4-dichlorophenol hydroxylase gene MIX-tfdB, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 5.

Another embodiment of the invention provides MIX-TfdB protein, which is characterized in that the amino acid sequence of the MIX-TfdB protein is shown in SEQ ID NO. 6.

Another embodiment of the invention provides a2, 4-dichlorophenol hydroxylase gene CTX3-tfdB, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.

Another embodiment of the invention provides a CTX3-TfdB protein, which is characterized in that the amino acid sequence of the CTX3-TfdB protein is shown in SEQ ID NO. 4.

Another embodiment of the present invention provides the use of the above gene MIX-tfdB or CTX3-tfdB for degrading Chlorophenols (CPs).

Another embodiment of the present invention provides the use of the above MIX-TfdB protein or CTX3-TfdB protein in degrading chlorophenol Compounds (CPs).

Another embodiment of the invention provides application of the genes MIX-tfdB or CTX3-tfdB in preparing recombinant proteins and/or engineering bacteria for degrading chlorophenols. The chlorophenol compound is preferably one or more of 2,4-DCP, 2,5-DCP, 3,4,5-TCP, 3,4-DCP, 2,3,4-TCP, 2,4,5-TCP and 2,3, 5-TCP.

The beneficial effects of the invention are as follows: the invention provides an artificially constructed 2,4-dichlorophenol hydroxylase gene MIX-tfdB and an expressed protein thereof, and CPs clearance experiments show that MIX-TfdB has higher enzyme activity on 2,4-DCP, 2,5-DCP and 3,4,5-TCP and higher removal rate; 3,4-DCP and 2,3,4-TCP have high removal rate although the enzyme activity is not high; in addition, the CTX3-tfdB gene and the expression protein thereof also show higher CPs removal rate.

Drawings

FIG. 1 is an east village harbor act Feng Dong river basin sample collection map; a (110.559836E-19.949968N), B (110.566490E-19.951913N), C (110.580206E-19.945682N); d (110.591480E-19.953493N); e (110.584549E-19.953397N).

FIG. 2 is a metagenomic DNA extraction assay; NM1: hind III DNA Marker, NM2:5kb DNA Marker,S01-S03 is soil sample metagenomic DNA sample, W01-W03 is water sample metagenomic DNA sample.

FIG. 3 is a non-redundant gene set length profile; and (3) injection: the abscissa indicates the length of the ORF sequence, and the ordinate indicates the number of sequences contained in the sequence.

FIG. 4 is a graph of the number of genes co-occurring in soil and water.

FIG. 5 is a functional histogram of Kegg annotated genes.

Figure 6 is a TfdB enzyme protein system evolution analysis (a), tfdB chimeric construction (b).

FIG. 7 is a diagram of SDS-electrophoresis identification, (a) CTX3-TfdB purified protein, (b) MIX-TfdB purified protein, (c) TfdB western blot assay, (a) 1: pET-30 a-to-BL 21 induction expression 2: CTX3-TfdB to BL21 did not induce 3: CTX3-TfdB to BL21 expression pellet 4: CTX3-TfdB to BL21 expression supernatant 5: CTX3-TfdB to BL21 expression purification 6:1×BSA 7:0.75×bsa8:0.5×bsa 9:0.25×bsa 10:0.1×BSA; (b) 1: pET-30 a-to-BL 21 induction expression 2: MIX-TfdB to BL21 did not induce 3: MIXT-fdB to BL21 expression pellet 4: MIX-TfdB to BL21 expression supernatant 5: MIX-TfdB to BL21 expression purification 6:1×BSA 7:0.75×bsa8:0.5×bsa 9:0.25 XBSA.

FIG. 8 is a graph of CTX3-TfdB enzyme activity assay.

FIG. 9 is a graph of MIX-TfdB enzyme activity assay.

FIG. 10 is a graph (a) showing the effect of different concentrations of FAD on TfdB enzyme activity, and a graph (b) showing the effect of different temperatures on TfdB.

Fig. 11 is a graph of the effect of pH on TfdB enzyme activity (a) and of the effect of ions on TfdB (b).

FIG. 12 is a graph of enzyme activities of different substrates, CTX3-TfdB (a), MIX-TfdB (b).

FIG. 13 is a chart of CPs removal rates, CTX3-TfdB (a), MIX-TfdB (b).

FIG. 14 is a map of plasmid pET-30a used in the present invention.

Detailed Description

In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.

The embodiment of the invention relates to a specific reagent, sample collection and general experimental operation method, which are as follows:

1. primary reagent and solution configuration

1) LB medium: pancreatic protein (Tryptone) 10g; 5g of yeast extract (yeast extract); 10g of sodium chloride (NaCl) and distilled water were added to a volume of 1L. The pH value is 7.2-7.4 (hand-held pH meter), and the solid culture medium is added with 1.5% agar. 121 ℃. And (5) sterilizing by high-pressure steam for 20 min.

2) TE buffer dissolution: 10mM Tris-HCl,1mM EDTA,pH =8.0;

3)J Buffer：0.1M Tris-HCl、0.1M EDTA、0.15M NaCl，pH＝8.0；

4) Restriction enzymes were purchased from Thermo company;

5) DNA Marker (No. B500347), DNA Marker lH, sanPrep column type PCR product purification kit (No. B518141), sanPrep column type plasmid DNA miniextraction kit (No. B518191), ready-to-use seamless cloning kit (No. B632219), isocitrate lyase (ICL) activity detection kit (No. D799839), ECL hypersensitive chemiluminescence kit (No. D6010139), 1xPBS buffer and SDS-PAGE Preparation kit (No. C631100) were all purchased from Shanghai Biotechnology;

6) The TureColor trichromatic pre-dye Marker (No. c 510010) contains 10 purified recombinant proteins (10, 17, 25, 33, 40, 53, 70, 95, 130, 180 kDa) in total from 10kDa to 180kDa, with the 70kDa band being orange, the 10kDa band being a mixture of blue and yellow bands, the remaining bands being blue. Suitable as a protein molecular weight standard for SDS-PAGE and Western.

7) 100mg/mL ampicillin (Amp): 1g of ampicillin powder is weighed and dissolved in 10mL of distilled water, filtered and sterilized by a sterile 0.22 mu m filter, and then packaged into 1.5mL of EP tube, stored at-20 ℃, and diluted 1000 times when in use;

8) 20mg/mL kanamycin (Kan): weighing 0.2g of kanamycin powder, dissolving in 10mL of distilled water, filtering and sterilizing by a filter dissolved in 0.22L in an ultra clean bench, subpackaging into 1.5mL of EP (European patent application) pipes, and preserving at-20 ℃ until the working concentration reaches 10ug/mL;

9) Agarose gel: 0.5g agarose was weighed out and dissolved in 50mL of 0.5 XTBE solution: weighing electrophoresis buffer solution, heating for 3min in a microwave oven at 300W, standing at normal temperature for about 15min, cooling to 50deg.C, sucking 3uL sample containing nucleic acid dye GVIII and 5uL DNA with a pipetting gun, mixing, and adding into sample hole;

10 0.5M IPTG: weighing 2.38g of IPTG (isopropyl-isopropyl M thiogalactoside) and dissolving in 20mL of distilled water, filtering and sterilizing by using a 0.22 filter, subpackaging into 1.5mL of EP pipe, and storing at-20 ℃;

11 5 XSDS-PAGE protein loading buffer: purchased from Shanghai assist, san Biotech Co.

12 5 XTris-glycine running buffer: tris base 15.1g,Glycin 94g,5g SDS, adding three distilled water for dissolution and then fixing the volume to 100mL. When in use, the three distilled water is diluted into 1 XTris-glycine electrophoresis buffer solution;

13 Coomassie brilliant blue staining solution): coomassie brilliant blue R250 g, methanol 180mL, acetic acid 40mL, add dd H ₂ Filtering the mixture into a brown bottle by using filter paper after fully and uniformly mixing, and preserving the mixture at normal temperature;

14 Fast decolorization liquid): taking 450mL of distilled water, 450mL of ethanol and 100mL of acetic acid, and fully and uniformly mixing;

15 Slow decolorizing liquid: 700mL of distilled water, 200mL of methanol and 100mL of acetic acid are measured and fully and uniformly mixed;

16 Transfer buffer 1000mL: glycine 2.9g,Tris base 5.8g,SDS 0.37g, 200mL of methanol and ddH2O triple distilled water were added to volume 1000mL.

17 PBST buffer: PBS containing 0.5mL/L Tween-20 (Tween-20).

18 Phosphate buffered saline (pH 7.4): 8g NaCl,0.2g KCl,1.44g Na ₂ HPO4 and 0.24 g KH ₂ PO4。

2. Sample collection

During the ebb period of 7 months in 2019, five sampling parties are set along the act Feng Dong river to collect water samples and soil samples, and the sampling parties cover the upstream, middle and downstream of the river (fig. 1). Firstly, a five-point sampling method is adopted to collect soil samples on each sampling side, sundries such as leaves, grass and the like covered on surface soil are removed, 4 repetitions are taken, 4 soil samples are obtained, and 20 soil samples are obtained for 5 sampling points. All soil samples were thoroughly mixed to remove debris and then stored in sterile plastic bags. A detailed record of the label was made, which was attached to the outside of the sample bag. The water samples were collected on the shore while soil samples were taken, a total of 20 water samples were taken at 5 sampling points, and all water samples (20 total L) were thoroughly mixed. And collecting microorganisms in the water body sample into a sterile centrifuge tube through centrifugation, and placing the collected soil and water body sample at-80 ℃ for storage after treatment.

3. Extraction of environmental (soil and water) microbial metagenomic DNA

Metagenomic DNA was extracted by direct lysis (He Jizheng et al 2012; zhao Yudong, zhou Jun, what, clear, 2012), the operations described below are described with respect to soil samples, and water sample metagenomic DNA extraction operations reference soil samples.

1) The soil was removed from-80℃and thawed at room temperature for about 30min, and 5g of the soil sample was weighed into a 100mL centrifuge tube.

2) To the sample, 14. 14mL DNA extraction buffer and 100. Mu.L proteinase K (10 mg/mL) were added, and the mixture was mixed.

3) Centrifuge tube was shaken horizontally at 37℃and 225rpm for 30min.

4) 2mL of 20% SDS was added, the mixture was cooled to room temperature by gently inverting the mixture several times every 15min in a 65℃water bath for 2 hours.

5) Centrifuge at 6000g for 10min at room temperature, collect supernatant and transfer to a new centrifuge tube.

6) To the pellet was added 4.5. 4.5mL DNA extraction buffer and 0.5mL 20% SDS, and gently vortexed until well mixed.

7) Water bath at 65 ℃ for 10min.

8) Centrifuging at 8000g for 6min at room temperature, collecting supernatant, and mixing with the supernatant.

9) The operations 6, 7, 8 were repeated once.

103 times of extraction with equal volumes of phenol: chloroform (1:1) is mixed, shaken well, centrifuged for 10min at 4 ℃ and 12,000g, mixed well and horizontally reversed for about 10 times.

11 Transfer to a new 100mL centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1), the level is reversed for about 10 times, and after being evenly mixed, the mixture is centrifuged for 10min at 4 ℃ and 12,000 g.

12 Transferring the supernatant again to a new centrifuge tube, adding 0.6 times volume of isopropanol, precipitating at room temperature for 1h, centrifuging at room temperature for 20min at 12,000g, and collecting the precipitate.

13 Washing the precipitate twice with pre-chilled 70% ethanol, gently mixing, and centrifuging at 4℃for 2min at 12,000 rpm/min.

14 100-500. Mu.L TE solution was added to the centrifuge tube, left overnight at 4℃and transferred to a 1.5mL centrifuge tube, centrifuged at 10,000rpm for 2min, and the supernatant was removed from the excess impurities.

4. Metagenomic sequencing

The extracted soil and water DNA samples were kept in dry ice, sent to Shanghai Meji Biotechnology (http:// www.majorbio.com) and double-ended by HiSeq 4000 platform (Inc., san Diego, calif., USA). Mainly comprises the following steps: preparing a library; breaking the genomic DNA into small fragments of several hundred bases (or less); terminal end adaptor; generating a DNA cluster; sequence information of the template DNA fragments is obtained by applying the principle of sequencing while synthesizing.

5. Microbial diversity analysis

The quality control is carried out on the original sequencing data of the macro genome in the environment of the mangrove forest in the east village harbor by using the software Seqprep, invalid bases in the original data are removed, and the reliability of data analysis and the accuracy of results are improved by high-quality data. ORF prediction was further performed. Selecting the gene with the length of the gene sequence being more than or equal to 100bp, and translating the gene into an amino acid sequence. All gene sequences predicted in the samples were clustered using CD-HIT software (default parameters: 90% identity,90% coverage). Constructing a non-redundant gene set explores commonalities and differences between different samples. And comparing the KEGG databases to obtain KEGG annotation profiles corresponding to the genes and carrying out statistics. The non-redundant gene set sequences were aligned to the KEGG gene database (GENES) using DIAMOND (parameters: blastp; E-value. Ltoreq.1E-5). The Venn diagram can be used for counting the number of species, functions or genes shared and unique among multiple groups or multiple samples, and can be used for clearly showing the composition similarity and overlapping condition among environmental samples.

Species, functions or gene abundance of each sample is counted on species, genes or functional layers, and dominant species, genes or functional compositions of communities are intuitively researched through a column diagram visualization method. Clustering is typically performed according to similarity of species (genes, functions) or abundance among samples, and the results are presented on a community hetmap, reflecting differences between different samples by color contrast. Inter-group significance difference test species between different groups (or samples) of microbial communities are analyzed according to the obtained reads abundance data to obtain species, functions or genes with obvious differences between the samples.

Table 1: software and parameters for each analysis of the study

6. Functional gene annotation screening

The annotation of gene functions depends on the gene structure and gene sequence, and functional information of the gene is obtained by comparing the gene sequence or protein sequence with a database, and finally, the functional annotation of the predicted encoding gene is performed (Li Tiezhu, etc., 2012). Based on the sequencing results, gene prediction was performed by software Glimmer 3.02 and Gene mark, ORF prediction was performed on predicted genomic components and possible encoding genes. And (3) constructing a blast sequence alignment database, and screening out genes possibly encoding specific functional proteins in the predicted genes through a blast alignment program (Zhang Enmin, marigold, dongzheng, 2009).

7. Plasmid DNA extraction

1) Plasmid DNA was extracted using a plasmid DNA miniextraction kit.

2) Single colonies were picked and placed into LB medium containing the corresponding antibiotics. Shaking table at 37 deg.C and 200rpm overnight;

3) 8,000rpm, centrifuging for 2min, pouring out the supernatant, and sucking out the redundant culture medium;

4) Adding 250 mu L buffer P1 into the bacterial precipitate, and blowing or vibrating until bacterial is thoroughly suspended;

5) Adding 250 mu L buffer P2, immediately and gently reversing and uniformly mixing, and standing at room temperature for 3min;

6) Adding 350 mu L buffer P3, immediately slightly inverting the mixture for about 5 times, and fully and uniformly mixing;

7) Centrifuging at 12,000rpm for 10min, transferring the supernatant to a new centrifuge tube, and discarding the precipitate;

8) Adding 0.5mL of isopropanol and uniformly mixing; centrifuging at 12,000rpm for 10min, carefully decanting the supernatant;

9) Adding 0.7mL of 70% ethanol solution, gently mixing, centrifuging for 2min, and removing the supernatant;

10 Repeating the 9 th step; centrifuging to volatilize ethanol;

11 Naturally drying for 20min, adding 40 μl TE solution, and storing at-20deg.C for subsequent test.

8. Gene PCR amplification

Primers (containing Xho1 and Nde1 cleavage sites) were designed to amplify the complete sequence of the gene by software primer 5.0 based on the predicted sequence of the gene of interest. PCR amplification of the DNA fragments was performed using the designed primers, and the PCR reaction system is shown in Table 2.3. Basic procedure for PCR amplification: pre-denaturing at 95 ℃ for 5min; carrying out denaturation at 95 ℃ for 1min; annealing at 55-70 ℃ for 1min; extending at 72 ℃ for reaction for 1min; then the reaction is carried out for 10min at 72 ℃; denaturation to extension was carried out for 30 cycles.

TABLE 2 PCR reaction System

9. Seamless cloning

1) Purifying the target gene PCR product and the carrier fragment after double enzyme digestion by using a purification kit;

2) Adding target genes, vector fragments and seamless cloned enzyme into the same EP tube according to an enzyme-linked reaction system;

3) An enzyme cleavage reaction system (Table 3), an enzyme-linked reaction system (Table 4);

4) Water bath at 50 ℃ for 30min;

5) Thawing HD5 alpha competent cells on ice;

6) Mixing enzyme-linked product and competent cells uniformly (inhibiting vortex), and placing on ice for 30min;

7) Placing in a metal water bath kettle at 42 ℃, and immediately transferring to ice for cooling for 2min after heat shock for 45 s;

8) Adding 450 mu L of LB liquid medium, and shaking at 200rpm at 37 ℃ for 1H;

9) Centrifuging at 12,000rpm and 4 ℃ for 2min, and removing 300 mu L of supernatant in a super clean bench;

10 Blowing and mixing the bacterial liquid in the EP pipe by a pipetting gun, uniformly coating the bacterial liquid on an LB plate containing corresponding antibiotics by a coating rod, and culturing the bacterial liquid in an incubator at 37 ℃ for 9-12H in an inverted manner;

11 Picking single colonies with equal size, and adding the single colonies into a liquid LB culture medium for overnight culture at 37 ℃;

12 Performing colony PCR, recombinant plasmid extraction PCR, enzyme digestion and other verification methods;

13 Screening positive transformants, and sequencing the transformants in Shanghai biological limited company;

14 DNA sequence analysis was performed using the Vector NTI Suite 11 software package from Invitrogen.

TABLE 3 cleavage reaction System

TABLE 4 enzyme-linked reaction system

10. Gene-induced heterologous expression

1) Inoculating the constructed expression engineering strain into LB culture medium containing corresponding antibiotics, and activating the expression strain overnight;

2) The following day was transferred to liquid LB medium containing the corresponding antibiotic in an amount of 1%, shaking culture at 37deg.C for about 2H to OD ₆₀₀ About 0.6, adding IPTG with proper concentration to 0.1-0.5-mM, and culturing 8H-20H;

3) No-load induction with IPTG as control, 2 parts of expression strain are transferred, one part is added with IPTG, and one part is used as negative control without IPTG;

4) Trying different temperatures, IPTG concentration and expression time, optimizing an expression system, and realizing soluble expression as far as possible;

5) Collecting thalli at 8,000g for 5min, washing 1-3 times with PBS solution, and washing 1 time with sterile water;

6) Adding a proper amount of PBS buffer solution, and treating for 5-20min in ultrasonic crushing until the liquid is clear (parameters: small probe power 20%, working 5s, interval 5 s);

7) Centrifuging to collect supernatant and precipitate respectively; the precipitate was washed 2 times with 1mL of PBS buffer, then 100 μl of PBS buffer was added to blow suspension;

8) Centrifuging the protein products of the expression strain and the reference strain for 5min at 10,000rpm, and collecting the supernatant and the precipitate respectively; washing 1-2 times with 1mL PBS buffer, mixing with 50. Mu.L PBS buffer, and performing SDS-electrophoresis analysis;

9) Bio-Rad densitometry scanning: scanning the protein gel using G800 and saving the picture;

10 Protein band SDS plots and their concentrations were analyzed using quality One software (Zhang et al 2012);

11 Expression verification is correct and can be expressed in a large amount, and the expression is induced by culturing in a 500mL culture flask.

11. Purification of expressed proteins

1) The obtained protein supernatant was mixed with Ni-NTA at a ratio of 1:4, mixing;

2) Placing the adsorption column on ice, and slowly and uniformly mixing with a 200rpm shaking table for 2H to fully combine the protein and Ni ions;

3) Opening a liquid outlet of the adsorption column to enable the leaching liquid to flow out, and collecting the leaching liquid;

4) Different concentrations of imidazole were configured: 20mM,50mM,70mM,130mM,170mM,202mM, 210mM, wash buffer with imidazole concentration of 10mM and Elutation buffer with imidazole concentration of 250mM are prepared; mixing the two reagents in proportion to obtain imidazole buffers with different concentrations;

5) 200. Mu.L of pre-chilled Wash buffer was added and the solution was placed on ice for 5min before releasing the Wash. Eluting for multiple times until the leaching solution is subjected to SDS-PAGE and has no protein band, and repeating the steps for one time;

6) Sequentially eluting with prepared imidazole concentration gradients, washing each gradient for at least 5 times, performing SDS-PAGE to obtain different elution effects of target proteins under different imidazole concentrations, and selecting the concentration with the best effect for the next time;

7) SDS-PAGE analysis of the eluate was performed and the eluate was placed at-20℃for further use.

12. SDS electrophoresis analysis

1) And (3) glue preparation: when the separating gel is prepared, adding a proper amount of n-butanol to eliminate bubbles, preparing concentrated gel after the separating gel is solidified, immediately inserting into a 1mm comb, and standing for 30min. Solidifying the gel with the concentrate for later use;

2) Sample treatment: mixing 16 mu L of protein sample with 4 mu L of 5 XSDS dye liquor buffer, and boiling for 5min;

3) Centrifuging at a low temperature of 4 ℃ for 12,000g and 5min, carefully sucking the supernatant into gel holes;

4) Using protein maker as a standard, 1%SDS running buffer (Tris15.1g, glycine 94g,SDS 5g,5,000mL H ₂ O)；

5) Performing constant voltage electrophoresis at 80V for 30min until sample protein is compressed and enters into the separation gel, regulating the voltage to 110V, and ending the constant voltage electrophoresis until bromophenol blue reaches the lower end of the gel;

6) Coomassie brilliant blue staining: carefully peeling the gel, washing, staining with coomassie brilliant blue for 30min, and placing on a horizontal oscillator;

7) Decoloring: washing the gel with distilled water after dyeing, adding 50mL of quick-release buffer solution, decoloring for 30min, adding slow-release buffer solution, and continuously shaking on a shaker until clear protein strips can be observed visually; and scanning by using a gel imager, analyzing and storing the picture.

13、Western blot

The protein is detected by using Western Blot, and the specific operation steps are as follows:

1) Electrophoresis: taking 16 mu L of protein sample, adding 4 mu L of 5 XSDS-PAGE loading Buffer, heating in boiling water for 5min, centrifuging at 12,000rpm for 5min, taking 16 mu L of supernatant, performing constant voltage electrophoresis at 80V for 45min, and performing constant voltage electrophoresis for 120min at a regulated voltage of 110V;

2) Transferring: cutting PVDF film consistent with the albumin glue, soaking in methanol solution for about 20s for activation, and placing the activated PVDF film and filter paper together into film transferring liquid for soaking. After electrophoresis, carefully taking out the protein gel, washing with distilled water after degumming, cutting off the area without protein, and soaking in the transfer film liquid; adopting a wet transfer method to transfer films according to the following sequence: the plate cathode, the sponge, the three layers of filter paper, the SDS-PAGE gel, the PVDF film, the three layers of filter paper and the plate anode are placed well and tightly sealed, and bubbles between the gel and the film and the filter paper are required to be removed during sealing, so that the influence on film transfer is avoided. Adding 1L of film transfer liquid into an electrophoresis tank, placing the electrophoresis device into an ice-water mixture, transferring film at 100V for 1H, and properly changing film transfer time according to the molecular mass of protein;

3) Closing: after the transfer, the PVDF membrane was transferred to a clean plastic box, washed with PBST or TBST at 100rpm with shaking for 10min, and repeated three times. Adding PBS (containing 1% BSA) blocking solution to block at normal temperature for 1H or at 4 ℃ for overnight after cleaning;

4) Antibody incubation: after the end of the blocking, discarding the blocking solution, washing for 10min by using PBST or TBST at 100rpm in an oscillating way, repeating for three times, and then adding primary antibody (1:1,000) into the blocking solution for incubation for 1H; adding a secondary antibody for incubation for 1H after washing the membrane;

5) Color development: after the incubation, discarding the incubation liquid, washing with PBST or TBST at 100rpm under shaking for 10min, repeating for three times, performing color development by using ECL super-sensitive chemiluminescence kit, uniformly mixing 1mL of the solution A of the chemiluminescence kit with 3 mu L of the solution B, uniformly spraying on the surface of the membrane by using a pipetting gun, sucking the redundant luminescence solution with filter paper after 1min, performing exposure imaging in a gel imager, and preserving.

14. Enzyme Activity assay

The enzyme activity was measured by ultraviolet spectrophotometry, and the enzyme activity of the enzyme was calculated indirectly by measuring the decrease in absorbance of NADPH at 340nm (Liuyang, 2011). The method for measuring the enzyme activity comprises the following steps:

1) Placing enzyme solution to be tested, 2,4-DCP mother liquor (prepared by acetone), NADPH mother liquor, phosphate buffer solution and ddH on ice in advance ₂ O；

2) To a 1.5mL EP tube, 200. Mu.L of 1mM 2,4-DCP mother liquor, and 400. Mu.L of 1mM NADPH mother liquor were added and left on ice for use;

3) After 50mM phosphate buffer (pH 7.5) was added thereto, 100. Mu.L of the enzyme solution was rapidly added thereto, and after mixing, the change in the ultraviolet absorbance at 340nm was measured at 25 ℃.

The units of TfdB enzyme activity are defined as: the amount of TfdB enzyme required to consume 1. Mu. Mol of NADPH in 1min was measured at 340nm wavelength at 25℃under standard conditions.

TfdB (U/mg) =ΔA/mol extinction coefficient×d×V inverse total×10 ⁹ (V sample×Cpr)/(T)

V inverse total: total volume of reaction, 1X 10 ^-3 L is; v sample: add sample volume, 0.1mL; NADH molar extinction coefficient, 6.22×10 ³ L/mol/cm; d:1mL quartz cuvette optical path, 1cm; cpr: sample protein concentration, mg/mL; v extraction: adding 1mL of the volume of the extracting solution; t: reaction time, 1min; unit conversion coefficient, 1 mol=10 ⁹ nmol。

The study carried out the measurement calculation on the TfdB enzyme dynamics data to further study the influence of different factors on the enzymatic efficiency. Ladder for setting temperatureThe temperature is15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃; the pH value is 4,5, 6, 7, 8, 9 and 10; metal ions have Mn ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、 Fe ²⁺ 、Zn ²⁺ 、K ⁺ 、Ca ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The TfdB activity under the above conditions was measured separately. The specific process is similar to that described in document 2.2.15. Because the enzyme has higher sequence and structural similarity with FAD dependent hydroxylase. We further studied the cofactors required for hydroxylase activity of CP homologs, adding 0.005mM FAD to the enzyme reaction system, and found that the rate of enzyme reaction was indeed increased, and then the effect of different concentrations of FAD on the enzyme activity of TfdB was measured to different extents.

15. Substrate-universality of TfdB enzyme

In the enzyme reaction system of the enzyme, different chlorophenol compound substrates are added, the types and physicochemical properties of the chlorophenol compounds are shown in table 2.6, and the enzyme activities of TfdB on various chlorophenol compounds are measured. High enzymatic activity towards CPs generally results in correspondingly high CP removal rates. And measuring the CPs removal rate after 60min of reaction by using an ultraviolet spectrometry method. The removal rate of CPs is the concentration of NADPH reducing amount divided by the amount of initial NADPH. In the TfdB enzyme reaction system, different CPs substrates are added, the initial NADPH value is measured, then the mixture is put into a water bath kettle at 37 ℃ to react for 60min, and the NADPH value is measured. And (5) sequentially calculating the removal rate of TfdB on different CPs substrates.

TABLE 5 TfdB substrate Compounds and physicochemical Properties thereof

16. Protein target molecule docking

TfdB target protein structures are obtained by using Swiss-Model online server homology modeling. After the related small molecules in the protein molecules are deleted by using the Pymol2.1 software, the TfdB protein molecules are imported into the Auto Dock Tools-1.5.6 software to delete water molecules, add hydrogen atoms and set atom types, and finally the TfdB protein molecules are saved as a pdbqt file. The 2,4-Dichlorophenol of the compound structure is from a PubCHem database, the energy of the 2,4-DCP is minimized through Chem3D, the 2,4-DCP is converted into a mol2 format, the three-dimensional structure of the 2,4-DCP is imported into Auto Dock Tools-1.5.6 to execute the same operation as the previous step, and finally the three-dimensional structure of the 2,4-DCP is saved as a pdbqt file. The treated 2,4-DCP is used as a molecular ligand, tfdB protein is used as a receptor, and the central position (x_center= -63.12, y_center= -34.424, z_center= -9.883) and the length, width and height of the Grid Box are determined according to the interaction of small molecules and targets are all set to be 40 multiplied by 40. Finally, batch molecular docking is carried out through Auto Dock, the result is further analyzed, and the combination effect of the 2,4-DCP and TfdB is visualized by using Pymol2.1 software. And evaluating the final docking structure based on the binding free energy.

Analyzing the action mode of the compound and the target protein to obtain the action condition of the compound and the protein residue, such as generated hydrogen bond action, pi-pi interaction, hydrophobic interaction and the like, and referring to the butt joint scoring of the compound, judging whether the compound to be screened has a certain activity action (An Song, 2020).

Note：Binding energy fuction：

ΔG _bind ＝C _lipo-lipo ∑f(γ _lr )+C _{hbond-neut-neut} ∑g(Δr)h(Δα)+C _{hbond-neut-charged} ∑g(Δr)h(Δα)+ C _{hbond-charged-charged} ∑g(Δr)h(Δα)+C _{max-metal-ion} ∑f(r _lm )+C _rotb H _rotb + C _{polar- phob} V _polar-phob +C _coul E _coul +C _vdw E _vdw +solvationterms

EXAMPLE 1 mangrove wetland water sample and soil sediment metagenomic DNA sequencing

The extracted metagenomic DNA was visualized by agarose gel electrophoresis as shown in FIG. 2. The band of the metagenomic DNA is more than or equal to 23kb, which indicates that the extracted DNA has better integrity. The quality detection of DNA concentration and purity by NanoDrop One, absorbance at A260/A280 was about 1.8, indicating that the metagenomic DNA was relatively pure with little contamination by proteins or other impurities. The results also indicate that the quality of the metagenomic DNA meets the sequencing requirements.

Metagenomic sequencing generates a large amount of data, with the total number of all sample genes before redundancy elimination being 10, 321, 205. The average number of raw readings in soil and water samples was 99, 317, 796 and 98, 984, 089, respectively. The quality control results of the metagenome sequences are shown in Table 6 and Table 7, and the total number of genes after redundancy removal is 36 and 590; the average number of quality control readings in soil and water samples after quality control was 98, 645, 909 and 97, 975, 913. From the data, it can be deduced that after the quality control of the sequences, the soil sample and the water sample account for about 99% of the original readings, and the soil sample and the water sample account for about 98% of the original base number. The quality control result of the sequence intuitively reflects that the sequencing quality of soil and water samples is good. By constructing the non-redundant gene set, the base sequence of the non-redundant gene set gene is obtained. The differences between the different samples can be reflected, and most of the sequences were found to be distributed between 200bp and 1,000bp, and a few were distributed at other lengths (FIG. 3). And processing the sequenced original data, removing the gene sequence with interference, and obtaining effective quality control data, thereby providing guarantee for the accuracy of subsequent bioinformatics analysis.

TABLE 6 statistical tables of number and length of genes before and after redundancy removal

TABLE 7 quality control statistics of metagenomic sequences

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Functional gene annotation screening

And (3) performing macro genome sequencing to generate a large amount of data, performing quality control to obtain high-quality control data, and performing analysis and statistics on macro genome data annotation results through comparison and annotation functions with a plurality of functional databases to obtain all gene function information in the east-to-west harbor mangrove environment. First, the genes in soil and water are compared, and the result shows that the specific genes in soil are 11,829, the specific genes in water are 14,003, and the common genes in water environment and soil environment are 10,758. The genetic profile between soil and water reveals that there is no independence between soil and water, and there is a relationship between the two habitats where microorganisms interact and are isolated from each other, as shown in fig. 4.

Annotating the macro genome data of the environment of the east village harbor mangrove forest by utilizing a KEGG database, and establishing a KEGG annotated gene function histogram. From FIG. 5, it can be seen that 21 different types of gene functions (the ratio is greater than 1%) are found out from the gene set by comparison with KEGG database and annotation analysis, wherein the four gene functions with the highest ratio are carbohydrate metabolism, amino acid metabolism, energy metabolism and nucleotide metabolism respectively. The obtained gene was compared with the KEGG gene database (Genes), and biological pathways of the corresponding functions in which the gene participated were obtained from the annotated gene analysis, and analysis of the pathways of the gene helped to further study the functions of the gene.

And finally obtaining the functional information of the genes through gene annotation. 4901 sequences are coded, and 17 genes which code for different enzyme functions are finally screened according to the functions of gene annotation, sequence integrity and the like, as shown in table 8. The 17 functional enzyme genes were further analyzed, and primers were further designed according to the sequences. The cloning and the expression of 7 genes are successfully completed by respectively carrying out seamless cloning and heterologous expression, and the gene sequence for coding TfdB is selected for subsequent experiments and analysis based on sequence analysis similarity, integrity and specificity of gene functions.

Table 8 identification of 17 functional enzyme genes

The preselected and screened target gene sequence information is clipped into primer 5.0, and the PCR primer sequence information is edited and synthesized by Shanghai Biotechnology, inc., see Table 9 in detail.

TABLE 9 primer information for target gene design

Note that: lower case letters in the F primer sequences represent homologous sequences at the upstream vector end of the 5 'end, and upper case letters represent forward amplified sequences specific for the 5' end gene; the lowercase letters in R indicate 5 'end downstream vector end homologous sequences, and the uppercase letters indicate 5' end gene-specific reverse amplification sequences.

Example 2TfdB sequence analysis and construction

The metagenome sequencing fragment is subjected to homology search in a Non-redundant UniProtKB/SwissProt sequences database by using a Blastp program provided by NCBI, found that a TfdB gene with the size of 939 bp codes 313 amino acid residues, has the greatest similarity (similarity of 50.32 percent and coverage of 98 percent) with the sequence of 2,4-dichlorophenol 6-monooxygenase (P27138.1) in the database, and is a novel 2,4-dichlorophenol hydroxylase gene, the inventor names the gene as TfdB-M153, the nucleotide sequence of which is shown as SEQ ID NO.1, and the coded protein (313 amino acids, the sequence of which is shown as SEQ ID NO. 2). By sequence comparison, the deletion of the C-terminal sequence of the TfdB-M153 gene is found, which is caused by the technology of metagenome sequencing. We have also found that a TfdB gene of 1749bp, which codes for 582 amino acid residues, is a novel 2,4-dichlorophenol hydroxylase gene, which has been named CTX3-tfdB by the inventor, and has a nucleotide sequence shown as SEQ ID NO.3, and a protein (582 amino acids, the sequence shown as SEQ ID NO. 4) encoded by the gene. Based on CTX3-tfdB gene, the deleted part of TfdB-M153 is complemented by utilizing a multi-sequence seamless cloning technology (figure 6), a novel TfdB gene with 1758bp is constructed, 585 amino acid residues are encoded, the similarity with Non-redundant UniProtKB/SwissProt sequences database 2,4-dichlorophen 6-monooxygenase (Q8KN28.3) is highest (the similarity is 44.21 percent and the coverage is 97 percent), the novel artificially constructed 2,4-dichlorophenol hydroxylase gene is named as MIX-tfdB by the inventor, the nucleotide sequence of the gene is shown as SEQ ID NO.5, and the encoded protein (585 amino acids are shown as SEQ ID NO. 6)

Example 3

1. TfdB gene expression and purification

To determine whether the recombinant protein obtained had TfdB bioactivity, two sets of enzyme activity control experiments were set up. The first group adds pET30a to BL21 recombinant protein in the enzyme reaction system to determine the enzyme activity of the enzyme reaction system, and the second group adds TfdB to BL21 recombinant protein in the enzyme reaction system to determine the enzyme activity of the enzyme reaction system, and analysis is carried out and a detection dynamics curve of TfdB enzyme activity is drawn. The two TfdB genes are expressed in E.coli BL21 (DE 3) as host cell.

The effect of different temperatures, different IPTG concentrations and different induction times on TfdB protein expression was explored separately, wherein the optimal induction concentration of recombinant CTX3-TfdB was 5 μl. The induction of 12H at 28℃resulted in FIG. 7a, which shows a slower protein expression rate when the temperature was lower, whereas the optimal induction concentration of recombinant MIX-TfdB was 5. Mu.L, and the induction of 8H at 16℃was used as the optimal temperature and optimal induction time for the expression of the foreign protein (FIG. 7 b). The recombinant protein N segment has 6 XHis, the recombinant protein is purified by adopting an affinity ion chromatography, and the protein concentration of the purified protein is measured by a Bradford method, so that the protein concentration of the purified protein is 0.138mg/mL and 0.079mg/mL respectively. The purified protein of interest was detected with an anti-His tag monoclonal antibody, which indicated that the protein of interest was a recombinant protein containing a 6 xhis tag (fig. 7 c).

2. TfdB enzyme Activity assay

Under the optimal expression conditions, the expressed proteins were subjected to enzyme activity assays. The enzyme assay during the removal of CPs was determined by monitoring the decrease in absorbance at 340nm after NADPH substrate dependent oxidation. To determine whether the recombinant protein obtained in this study had the biological activity of the enzyme, the OD size was not changed substantially within ten minutes in the reaction system without TfdB addition, the reaction began to proceed after TfdB addition, the OD value began to decrease, and the FAD concentration of 0.005mM had some promotion of the TfdB enzyme activity. The total time for the whole reaction test was 10min (FIG. 8).

The decrease in NADPH was measured at 340nm absorbance to reflect TfdB enzyme activity. Wherein the absorbance of CTX3-TfdB enzyme activity reaction for 1min is reduced by 0.145, and according to the protein concentration of 0.138mg/mL, the formula TfdB (U/mg) =DeltaA/molar extinction coefficient x d x V x 10 is calculated ⁹ ÷(V×Cpr)÷T＝1.68 μmol/(mg·min)。

Absorbance decrease of 0.086 in 1min in MIX-TfdB enzymatic activity reaction, according to protein concentration of 0.079mg/mL, calculated as: tfdB (U/mg) =ΔA/mol extinction coefficient×d×V×10 ⁹ ÷(V×Cpr)÷T＝1.75 μmol/(mg·min)

When the TfdB sequence is analyzed, the enzyme is possibly a FAD dependent enzyme, the enzyme reaction system is added with 0.005mM FAD, the enzyme reaction rate is indeed increased, then the TfdB enzyme activity is analyzed by different concentrations of FAD, and the TfdB enzyme activity is promoted to different degrees by different concentrations of FAD, wherein the enzyme activity promotion by CTX3-TfdB is higher by the added concentration of 0.005mM FAD. The enzyme activity promoting effect on MIX-TfdB was not evident by the addition of different concentrations of FAD (fig. 10 a).

In order to explore the influence of various factors on TfdB enzyme reaction, experimental analysis is carried out on the optimal pH value, the optimal temperature, the optimal metal ions and the optimal cofactors. The optimum temperature of the enzyme was determined in the temperature range of 0℃to 55 ℃. CTX3-TfdB enzyme has highest activity at 20 ℃ and higher catalytic activity at 20-40 ℃, MIX-TfdB enzyme has highest activity at 40 ℃ (figure 10 b), and the highest optimal enzyme activity can be related to the special habitat of mangrove.

The enzyme activity is higher under neutral conditions, whereas the stability of the enzyme is worse under other conditions, when the pH is below 6.0 or above 8.0, the CTX3-TfdB enzyme activity is lower. The CTX3-TfdB enzyme has optimal enzyme activity at pH 7.4. The optimum pH for MIX-TfdB was 6.0. The MIX-TfdB enzyme has better stability in a wider range (figure 11 a), and the results show that the MIX-TfdB enzyme can play a normal catalytic role under weak acidic condition or neutral condition. This feature may be related to the strong acidic habitat conditions of mangrove.

The enzyme activity was then determined by adding different ions to the enzyme solution, and the results showed (FIG. 11 b) Mg ²⁺ Has promoting effect on CTX3-TfdB enzyme activity, and Cu ²⁺ 、Zn ²⁺ Has inhibiting effect on CTX3-TfdB enzyme activity. Mg of ²⁺ 、Ca ²⁺ Has inhibitory effect on MIX-TfdB enzyme activity. Fe (Fe) ²⁺ It is presumed that the CTX3-TfdB enzyme activity is promoted by binding of a metal ion to a certain group of the enzyme molecule, resulting in a decrease or loss of the enzyme activity.

3. Enzyme substrate specificity assay

The substrate specificity of the enzyme was determined using chlorophenol substrates of different structures. The present study uses ultraviolet spectrophotometry to detect TfdB enzyme activity. Under the condition of 37 ℃, the reaction rates of different chlorophenol compound substrates are catalyzed by enzymes, and the result proves that TfdB has different degradation activities on the selected chlorophenol compound substrates, and the addition of the FAD with proper concentration can increase the reaction rate, so that the addition of the FAD has a certain pushing effect on the reaction of the TfdB for catalyzing the chlorophenol compound. Of the selected substrates, both TfdB had the highest activity towards 2,4-dichlorophenol, while CTX3-TfdB had higher activity towards 2,3, 5-trichlorophenol, 2, 5-dichlorophenol, 3, 5-trichlorophenol, 3,4, 5-tetrachlorophenol. MIX-TfdB has high activity on 2, 3-dichlorophenol, 2, 5-dichlorophenol, 3, 4-dichlorophenol, 3, 5-trichlorophenol and 3,4, 5-tetrachlorophenol. The addition of FAD at a concentration of 0.005mM can effectively increase the reaction rate of TfdB. However, CTX3-TfdB and MIX-TfdB do not react to the addition of FAD to the same extent. For example, the addition of 0.005mM FAD increases CTX3-TfdB to about 30% of 3, 4-dichlorophenol activity. However, the addition of 0.005mM FAD did not significantly increase MIX-TfdB to 3, 4-dichlorophenol activity. It can be seen that the promotion of the two enzymes by FAD is not the same when the substrates are identical. In general, both enzymes have some activity on the chlorophenol substrate tested. Therefore, tfdB is a very novel and potentially an enzyme, which is beneficial in being able to convert more aromatics afterwards.

4. TfdB enzyme to CPs removal rate

The removal rate of CPs should be confirmed by high performance liquid chromatography, and also by measuring the UV-detected NADPH consumption. The present study indirectly reflects the removal rate of CPs by detecting the decrease in NADPH using an ultra-micro spectrophotometer. The results indicate that high enzymatic activity of CPs generally results in correspondingly high removal rates of CPs. As shown in fig. 13 a. CTX3-TfdB has higher enzyme activity on 2,4-DCP, 2,5-DCP and 2,3,5-TCP, and has higher removal rate. However, 3,5-DCP and 2,3,4-TCP have high removal rates but their enzymatic activities are not high. As shown in FIG. 13b, MIX-TfdB has higher enzyme activity on 2,4-DCP, 2,5-DCP, 3,4,5-TCP and higher removal rate. However, 3,4-DCP and 2,3,4-TCP have high removal rates but their enzymatic activities are not high. The results indicate that the high enzymatic activity of CPs generally results in correspondingly high removal rates of CPs, but with exceptions, may have a certain relationship with the structure of the CPs.

Depending on the nature of the enzyme, the reaction catalyzed by a particular hydroxylase requires FAD as a cofactor to stimulate its substrate. TfdB has high sequence and structural similarity with FAD-dependent hydroxylase and contains FAD as a prosthetic group. TfdB catalyzes FAD-dependent oxidative hydroxylation of 2,4-DCP and its homologs. The requirement of FAD for hydroxylase activity and removal of 19 CP homologs was studied to determine if FAD is a necessary cofactor for the enzyme. The results indicate that 0.005mMb FAD has different degrees of promotion for different CPs.

Claims (8)

1. The 2,4-dichlorophenol hydroxylase gene MIX-tfdB is characterized in that the nucleotide sequence is shown in SEQ ID NO. 5.

2. A MIX-TfdB protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 6.

3. A2, 4-dichlorophenol hydroxylase gene CTX3-tfdB is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.

4. A CTX3-TfdB protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 4.

5. Use of the gene MIX-tfdB according to claim 1 or the gene CTX3-tfdB according to claim 3 for degrading Chlorophenols (CPs).

6. Use of MIX-TfdB protein according to claim 2 or CTX3-TfdB protein according to claim 4 for degrading Chlorophenols (CPs).

7. Use of the gene MIX-tfdB according to claim 1 or the CTX3-tfdB according to claim 3 for the preparation of recombinant proteins and/or engineering bacteria for degrading chlorophenols.

8. The use according to claim 7, wherein the chlorophenol compound is selected from one or more of 2,4-DCP, 2,5-DCP, 3,4,5-TCP, 3,4-DCP, 2,3,4-TCP, 2,4,5-TCP, 2,3, 5-TCP.

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