Oxidase gene bpfA as well as encoded protein and application thereof

Abstract

The invention discloses an oxidase gene bpfA, a protein coded by the oxidase gene bpfA and application of the oxidase gene bpfA. The overall length of the oxidase gene bpfA is 1599 bp, the sequence of the oxidase gene bpfA is shown as SEQ ID NO.1, and the encoded product oxidase BpfA contains 532 amino acids, and the sequence of the oxidase gene bpfA is shown as SEQ ID NO.2. The BpfA can be used for degrading bisphenol F (BPF). The oxidase BpfA can be used for degrading BPF residues in water and soil, and has very important theoretical and application values.

Description

Technical Field

[0001] The present invention belongs to the fields of environmental microorganisms and agriculture, and relates to an oxidase gene bpfA and a protein encoded thereby and applications thereof, and in particular to an oxidase gene bpfA for degrading bisphenol F (BPF) and applications thereof. Background Art

[0002] Bisphenols (BPs) are important chemical raw materials for the synthesis of polymers and are widely used in various consumer products. Bisphenol A (BPA) is the most representative of these compounds. However, toxicological studies have shown that BPA has endocrine disrupting effects and may cause a variety of health problems, including cancer, reproductive disorders, and obesity. In recent years, with increasing concern about the potential hazards of BPA, alternatives have gradually emerged, including bisphenol F (BPF), bisphenol B (BPB), bisphenol S (BPS), and bisphenol AF (BPAF). However, toxicological studies of these alternatives have found that their biological toxicity and environmental behavior are similar to those of BPA, or even more complex.

[0003] As a major BPA substitute, BPF is widely used in the production of polycarbonate plastics, epoxy resins, coatings and electronic products. Due to its high chemical stability, its persistence and mobility in the environment have led to increasingly prominent residue problems. Studies have shown that BPF can enter water bodies, soil and the atmospheric environment through industrial wastewater discharge, aging of plastic products and landfill infiltration, and has been widely detected in global water environments. Because BPF is lipophilic, it can be exposed to the water and food phases and enriched in organisms, and transferred along the food chain, affecting human health through bioaccumulation. Therefore, it is necessary to develop effective BPF degradation treatment methods.

[0004] Obtaining BPF degradation genes has the following applications in the treatment of BPF residues in the environment: (1) Eliminating BPF residues in soil and water; (2) Constructing genetically engineered strains through genetic research and then studying their enzymatic properties is of great significance for the remediation of BPF pollution. Therefore, degradation genes have very important theoretical and practical value in eliminating BPF residues. Summary of the Invention

[0005] The purpose of the present invention is to provide a BPF oxidase gene bpfA;

[0006] Another object of the present invention is to provide the protein BpfA encoded by the gene;

[0007] Another object of the present invention is to provide applications of the gene;

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] An oxidase gene bpfA, whose nucleotide sequence is SEQ ID NO.1.

[0010] The target gene was found by combining multiple protein purification and genome sequencing methods. First, the BPF oxidase from the crude enzyme solution of Microbacterium sp. F2 was gradually purified using ammonium sulfate precipitation, DEAE-Sepharose Fast Flow ion exchange chromatography, Q-Sepharose Fast Flow ion exchange chromatography, and Sephadex-200 gel chromatography. Finally, after Sephadex-200 gel chromatography, the BPF-degrading activity was still present, as shown by the polyacrylamide gel electrophoresis pattern (see Figure 3 ) and purified a single protein, preliminarily determining the BPF oxidase protein size of this strain to be approximately 58.8 kDa. This band was sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for peptide fingerprint analysis. The genome of strain F2 was then extracted using a high-salt method, and DNA samples were sent to Lingen Biotechnology for genome sequencing. Homology analysis of the peptide sequence with the amino acid sequence of strain F2 revealed an 83% similarity with ORF1733 in scaffold 2. Therefore, ORF1733 was analyzed, and the corresponding encoding gene was named bpfA.

[0011] The protein BpfA encoded by the oxidase gene bpfA, whose amino acid sequence is SEQ ID NO.2, was commissioned to Beijing Qingke Biotechnology Co., Ltd. to synthesize the oxidase gene bpfA.

[0012] A recombinant expression vector containing the oxidase gene bpfA.

[0013] The recombinant expression vector is preferably obtained by connecting the oxidase gene bpfA to the NdeI and XhoI sites of pET-29a(+).

[0014] The genetically engineered bacteria containing the oxidase gene bpfA, the genetically engineered strain preferably uses Escherichia coli BL21 (DE3) as the starting strain.

[0015] The oxidase gene bpfA is used in the degradation of bisphenol F (BPF).

[0016] The recombinant expression vector containing the oxidase gene bpfA is used to degrade BPF.

[0017] The application of the oxidase protein BpfA in degrading BPF.

[0018] The oxidase protein BpfA is used in preparing a reagent for removing BPF from water or soil or in removing BPF from soil or water.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The present invention isolated a strain capable of degrading BPF from an enriched solution, designated Microbacterium sp. F2. Based on this, the present invention successfully cloned the oxidase gene bpfA from strain F2 using a combination of multiple protein purification techniques and strain genome sequencing. Online amino acid sequence analysis using blastp and homology comparison in the NCBI (UniProtKnowledgeBase / SwissProt databases) revealed that the gene is novel, with a full length (from start codon to stop codon) of 1599 base pairs (bp) encoding 532 amino acids.

[0021] 2. The oxidase BpfA provided by the present invention can completely degrade 20 mg / L of BPF within 1 hour. In addition, BpfA can also be used to construct a genetically engineered strain of BPF to remove BPF residues in water and soil, which has very important theoretical and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 HPLC spectrum of BPF degradation by strain F2 and MS spectrum of metabolites;

[0023] A: BPF standard sample; B: HPLC spectrum of BPF degradation by F2; C: MS spectrum of BPF; D: MS spectrum of 4,4′-dihydroxybenzophenone (DHBP).

[0024] Figure 2 Diagram of the cloning strategy for the oxidase gene bpfA;

[0025] Figure 3 The different steps of purification of oxidase BpfA from strain F2 were detected by SDS-PAGE;

[0026] Lane 1: protein marker; Lane 2: crude enzyme solution of strain F2; Lane 3: purification by ammonium sulfate precipitation; Lane 4: purification by DEAE-Sepharose anion exchange chromatography; Lane 5: purification by Q-Sepharose cation exchange chromatography; Lane 6: purification by Superdex G-200 column.

[0027] Figure 4 Diagram of the expression strategy of the oxidase gene bpfA in BL21 (pET-29a).

[0028] Figure 5 Electrophoresis pattern of oxidase BpfA protein;

[0029] Lane 1 is a protein marker; lane 2 is the purified oxidase BpfA protein.

[0030] Figure 6 HPLC spectrum of BPF degradation catalyzed by oxidase BpfA and MS spectrum of metabolites;

[0031] A: HPLC spectrum of BPF degradation by BpfA; B: MS spectrum of BPF; C: MS spectrum of DHBP. DETAILED DESCRIPTION

[0032] Example 1 Isolation and screening of BPF-degrading strain F2

[0033] 1.1 Enrichment, acclimation and isolation of BPF-degrading strain F2

[0034] Samples were collected from hospital medical wastewater, and a strain capable of degrading BPF was isolated through enrichment culture. 5 mL of sample was added to 100 mL of MSM liquid culture medium containing 30 mg / L BPF. Culture was maintained at 30°C with shaking at 180 rpm. Every other week, a 5% inoculum was transferred to fresh MSM liquid culture medium containing 30 mg / L BPF. The strain was subcultured three times.

[0035] Basal salt medium (MSM) formulation: K₂HPO₄·3H₂O 1.5 g; KH₂PO₄ 0.5 g; NH₄Cl 1.0 g; NaCl 1.0 g; MgSO₄·7H₂O 0.2 g. Add deionized water to 1 L. Add 15.0 g agar per liter of solid medium.

[0036] 1.2 Purification, screening and identification of degradation strains

[0037] High performance liquid chromatography was used to detect whether BPF in the third generation enriched solution was degraded and whether metabolites were generated. -1 to 10 -7The strains were plated with multiple dilutions of 500 μg / mL onto MSM solid medium supplemented with 30 mg / L BPF and incubated in a 30°C incubator for one week. Single colonies of varying morphology grown on the plates were selected and placed into LB liquid test tubes. After incubation at 30°C, 180 rpm, and 3 weeks in a shaker, 1 mL of the bacterial solution was transferred to a sterile 1.5 mL centrifuge tube and centrifuged at 6,000 × g for 3 minutes. The strains were washed three times with sterile MSM liquid medium and then transferred to MSM liquid medium supplemented with 30 mg / L BPF. The strains were shaken and incubated at 30°C, 180 rpm, for one week. HPLC was used to identify the BPF-degrading properties of the individual colonies.

[0038] A BPF-degrading strain, named F2, was isolated through enrichment, acclimation, and screening. After four days of growth on LB solid plates, strain F2 produced yellow, circular colonies with moist surfaces and regular edges. Strain F2 is a Gram-positive bacterium.

[0039] Phylogenetic analysis of 16S rRNA gene sequences: Total DNA of the strain was used as a template for PCR amplification using universal primers 27F / 1492R for 16S rRNA gene. The primer 27F was 5′-AGAGTTTGATCCTGGCTCAG-3′, and the primer 1492R was 5′-TACGGCTACCTTGTTACGACTT-3′.

[0040] Amplification reaction system (50 μL):

[0041]

[0042] The procedure used is as follows:

[0043]

[0044] The PCR products were detected by electrophoresis on a 1.0% agarose gel. After recovery, the PCR products were ligated with the pMD19-T vector and transformed into E. coli DH5α competent cells. After cultivation, positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rRNA gene obtained after sequencing was compared with the EZtaxon database (https: / / www.ezbiocloud.net / ). The strain F2 and Microbacterium saccharophilum K-1 T The sequence similarity with that of the strain F2 was 99.38% (GenBank accession number: AB736273), and the strain F2 was identified as Microbacterium sp.

[0045] 1.3 Detection of BPF concentration

[0046] UV spectrophotometer detection: Take 1 mL of the sample to be tested, add an equal volume of methanol to dissolve it, centrifuge at 12,000 × g for 3 minutes, scan the upper liquid within 200-400 nm, and detect the change in BPF content by the change in the characteristic absorption peak and response value at 270 nm.

[0047] HPLC detection: Take 1 mL of the sample to be tested, add an equal volume of methanol to dissolve it, centrifuge at 12,000 × g for 3 minutes, filter the supernatant through an organic phase filter, and use high-performance liquid chromatography (HPLC) to determine the BPF content. HPLC detection conditions: mobile phase: 0.1% acetic acid: 100% methanol = 4:6; column temperature: 40°C; mobile phase flow rate: 0.8 mL / min; detection wavelength: 270 nm, injection volume: 20 μL.

[0048] 1.4 Degradation characteristics of the degradation strain F2 and analysis of degradation products

[0049] Degradation characteristics: strain F2 was inoculated into 100 mL LB liquid medium at a 1% inoculum, placed in a constant temperature shaker at 30°C and 180 rpm, and samples were taken every 4 hours to measure OD 600 The strain was inoculated into LB liquid medium at a 1% inoculum and cultured at 30°C, 180 rpm constant temperature shaker until the logarithmic growth phase. The cells were collected by centrifugation at 6,000 × g for 3 minutes, resuspended in MSM liquid medium, and centrifuged twice. The cells were washed thoroughly and resuspended in MSM liquid medium until the OD 600 The final OD value of strain F2 was about 1.0, which was used as the seed solution of strain. Strain F2 was added to the MSM liquid medium containing 30 mg / L BPF, and the final OD value of strain F2 was 600 The culture was incubated in a constant temperature shaker at 0.2, 30°C, and 180 rpm. Samples were taken every 2 hours, and the changes in BPF content during the degradation of BPF by strain F2 were determined using HPLC.

[0050] The experimental results showed that strain F2 could degrade BPF and could degrade 20 mg / L BPF after 25 h of culture. HPLC-MS analysis showed that BPF was degraded to produce DHBP ( Figure 1 ).

[0051] Example 2 Cloning and functional verification of BPF oxidase gene (strategy diagram see Figure 2 )

[0052] 2.1 Sequencing analysis of total bacterial genomic DNA

[0053] 2.1.1 Extraction of total bacterial genomic DNA

[0054] The strain F2 was cultured in LB liquid and the cells were collected by centrifugation to verify the strain's ability to degrade BPF. The total genomic DNA of the strain F2 was extracted using a high salt combined with CTAB method and stored at -20°C.

[0055] 2.1.2 Genome draft sequencing and result analysis

[0056] The extracted total DNA was sequenced by Lingen Biotechnology Co., Ltd.: 1. Using Illumina TruSeq TM 1. Construct the library using the NanoDNA Sample Prep Kit; 2. Analyze the sequencing data.

[0057] 2.1.3 Analysis of draft genome sequencing results

[0058] Genome sequencing results: The draft genome of strain F2 was 3,209,201 bp in size, with 26 scaffolds and a G+C content of 66.91%.

[0059] 2.2 Protein purification of crude enzyme solution from strain F2 cell disruption

[0060] 2.2.1 Preparation of cell lysis solution

[0061] Strain F2 was inoculated into LB medium and cultured in a shaking incubator at 30°C until OD 600 The cell density was approximately 2.0. The cells were collected by centrifugation at 6,000 × g for 3 minutes, resuspended in Tris-HCl buffer (pH 7.4), washed twice, and resuspended in 15 mL of Tris-HCl buffer. The cells were disrupted using an ultrasonic disruptor in an ice bath. After disruption, the cells were centrifuged at 12,000 × g for 20 minutes at 4°C. The supernatant was collected and filtered through a 0.22 μm water filter to remove the cells and debris. The crude enzyme solution of strain F2 was obtained and stored at 4°C for subsequent purification.

[0062] 2.2.2 Determination of crude enzyme activity of strains

[0063] 1mL enzyme activity reaction system: Add 27μL of strain F2 crude enzyme solution and 3μL of BPF (30mg / L) to 970μL of Tris-HCl buffer (pH 7.4). Incubate in a 30°C water bath for 1 hour. Terminate the reaction by adding an equal volume of methanol and boiling for 10 minutes. Determine the BPF concentration by HPLC. Definition of one unit (U): In this assay, one unit (U) is defined as the amount of enzyme (mg) required to reduce 1μmol of BPF per minute.

[0064] The enzyme activity determination experiment showed that the specific enzyme activity of the crude enzyme solution of strain F2 in degrading BPF can reach 14.93U / mg.

[0065] 2.2.3 Ammonium sulfate fractional precipitation

[0066] The crude enzyme solution of F2 was subjected to graded precipitation at sulfuric acid saturations of 0-20%, 20-40%, 40-60%, 60-80%, and 80-100%. The volume of the crude enzyme solution was calculated, and the mass of ammonium sulfate was calculated based on the volume. The crude enzyme solution of F2 was placed in a conical flask with a built-in magnetic stirrer rotor. The conical flask was placed in an ice bath beaker. The magnetic stirrer was turned on to a constant speed. Ammonium sulfate was slowly added to the saturation of 20%. The solution was centrifuged at 4°C and 12,000×g for 30 minutes. The precipitate was resuspended in an appropriate amount of 20mM Tris-HCl buffer (pH 7.4) and dissolved on ice. The supernatant was collected and the above steps were repeated until the ammonium sulfate saturation of the paper reached 100%. After the precipitate of each component was completely dissolved, it was dialyzed overnight against 20mM Tris-HCl buffer (pH 7.4) at 4°C using a dialysis bag with a molecular weight cutoff of 10 kDa. After the dialyzed enzyme solution is subjected to protein quantification and enzyme activity detection, the activity recovery (%) and protein content (%) of each level of precipitation are calculated, and the fraction with high enzyme activity is selected for further purification.

[0067] 2.2.4DEAE-Sepharose fast flow ion column chromatography

[0068] Equilibrate a DEAE-Sepharose fast flow anion column with 20mM Tris-HCl (pH 7.4) buffer. Slowly add the enzyme solution with the highest activity, collected in the previous step, to the column. Elute with 20mM Tris-HCl (pH 7.4) buffer, then perform a gradient elution using 20mM Tris-HCl (pH 7.4) buffer containing varying concentrations of NaCl (0.20M, 0.25M, 0.3M, and 0.35M), collecting 3 mL of each solution in a tube. Assay the enzyme activity of the collected enzyme solution, pool the enzyme solutions with the highest activity, dialyze overnight, and finally concentrate by ultrafiltration using a 10kDa ultrafiltration tube at 4°C and 3,000×g.

[0069] 2.2.5 Q-Sepharose fast flow ion column chromatography

[0070] A Q-Sepharose fast flow anion column was equilibrated with 20mM Tris-HCl (pH 7.4) buffer. The concentrated enzyme solution from the previous step was added to the column and eluted with 20mM Tris-HCl (pH 7.4) buffer. A gradient elution was then performed using 20mM Tris-HCl (pH 7.4) buffer containing varying concentrations of NaCl (0.20M, 0.25M, 0.3M, and 0.35M). Each 3mL fraction was collected into a tube and enzyme activity was measured. The active enzyme solution was dialyzed overnight and finally concentrated by ultrafiltration using a 10kDa ultrafiltration tube at 4°C and 3,000×g.

[0071] 2.2.6 Superdex-200 gel chromatography

[0072] First, equilibrate a Superdex G-200 molecular sieve column with 20 mM Tris HCl (pH 7.4) buffer containing 0.1 M NaCl for 2–3 column volumes. Slowly inject the ultrafiltered enzyme solution into the injection valve to begin elution at a flow rate of 0.4 mL / min, collecting 0.5 mL of solution in a tube. After elution is complete, rinse the column and store it in 20% ethanol until ready for use. A suitable amount of the collected enzyme solution is then collected for enzyme activity assay.

[0073] 2.2.7 Polyacrylamide gel electrophoresis (SDS-PAGE)

[0074] Take 30 μL of the active enzyme solution after each level of purification, add 10 μL of 4×SDS-PAGE Loading Buffer, boil in boiling water for 10 minutes, take out and cool to room temperature, add 20 μL of sample volume to the SDS-PAGE loading wells in sequence, turn on the power, and wait until bromophenol blue reaches the bottom of the separation gel, and the electrophoresis is completed; place the gel in a fresh-keeping box, pour in an appropriate amount of Coomassie Brilliant Blue R-250 staining solution, and stain for 30 minutes; pour out the staining solution, add an appropriate amount of destaining solution, and slowly shake on a shaker for 8 to 10 hours to destain, during which time the destaining solution is changed 2 to 3 times, until the bands are clearly visible.

[0075] 2.2.8 Oxidase peptide fingerprint analysis

[0076] The target protein was detected by SDS-PAGE ( Figure 3), the band containing the target protein was cut and sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for LC-MS / MS protein spectrum identification. The peptide sequence was compared with the amino acid sequence in the genome of strain F2 for homology analysis, and an ORF annotated as an oxidase was found, named BpfA, with 532 amino acids and a coding nucleotide length of 1599 bp. The nucleotide sequence of the coding gene bpfA is shown in SEQ ID NO.1, and the amino acid sequence of BpfA is shown in SEQ ID NO.2.

[0077] Example 3 Oxidase gene bpfA expression and verification (strategy diagram see Figure 4 )

[0078] 3.1 Synthesis of expression vector for BPF oxidase gene bpfA

[0079]

[0080] The bpfA gene vector pET-bpfA was synthesized by Beijing Qingke Biotechnology Co., Ltd., wherein the nucleotide sequence of the bpfA gene is shown in SEQ ID NO.1.

[0081] 3.2 Construction and verification of expression strains

[0082] Transform the constructed expression vector into the E. coli BL21(DE3) expression strain. Select transformants and transfer them to test tubes containing LB liquid containing 50 mg / L Km and culture them in a shaker at 37°C and 180 rpm. PCR was used to verify the correctness of the resulting strain. The resulting positive clone was named E. coli BL21(DE3)-bpfA.

[0083] 3.3 Induced expression and purification of expression vector

[0084] Select a single colony of E. coli BL21 (DE3)-bpfA and place it in LB liquid medium containing Km (50 mg / L) and culture it at 37°C and 180 rpm for about 8 hours. When the bacteria grow to the logarithmic phase, take 1 mL of the bacterial solution and inoculate it into 100 mL of LB liquid medium and culture it at 37°C and 180 rpm in a shaker until the OD reaches 0. 600The pH value was 0.4 to 0.6, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM. The culture was induced at 16°C and 180 rpm for 10 hours. After the induction, the bacterial suspension was centrifuged at 12,000 × g for 2 minutes, the bacteria were retained, washed with Tris-HCl buffer and resuspended. The bacterial suspension was placed in an ice-water mixture and the cells were broken using an ultrasonic disruptor. The crude enzyme solution after the crushing was stored at 4°C, centrifuged at 12,000 × g for 20 minutes, and the upper enzyme solution was retained. After filtering with a 0.22 μm aqueous phase filter, the crude enzyme solution was stored in a refrigerator at 4°C.

[0085] The histidine tag on the pET-29a(+) vector was used as a marker and Ni 2+ The target protein BpfA was isolated and purified using a -NTA affinity chromatography column, and the eluate with the highest concentration was dialyzed overnight. The purification effect was detected by SDS-PAGE protein electrophoresis, and the band size was consistent with the theoretically predicted size (58.8kDa) ( Figure 5 ).

[0086] 3.4BpfA activity assay

[0087] A standard reaction system (1 mL) for BpfA oxidase was established: 30 mg / L BPF, 18.88 μg BpfA oxidase, and 20 mM Tris-HCl (pH 7.4) buffer to 1 mL. The enzymatic reaction system was incubated at 30°C for 1 hour. The reaction was terminated by adding an equal volume of methanol and boiling for 10 minutes. The solution was filtered and the BPF content was determined by HPLC during the reaction. The BpfA oxidase activity was assayed using this standard enzyme reaction system.

[0088] Definition of Enzyme Activity Unit (U): In this experiment, 1 U is defined as the amount of enzyme (mg) required to reduce 1 μmol of substrate per minute. Degradation experiments demonstrated that purified BpfA could degrade 20.0 ppm of BPF within 1 hour. Enzyme assays revealed that the specific activity of BpfA against BPF was 14.93 U / mg.

[0089] 3.5 Identification of metabolites

[0090] The products after the enzymatic reaction were detected and identified by HPLC technology.

[0091] HPLC conditions for BPF: mobile phase: 0.1% acetic acid: 100% methanol = 4:6; column temperature: 40°C; mobile phase flow rate: 0.8 mL / min; detection wavelength: 270 nm, injection volume: 20 μL.

[0092] HPLC analysis showed that BpfA could oxidize BPF and generate the corresponding oxidation product DHBP ( Figure 6 )。

Claims

1. An oxidase gene bpfA, characterized in that The nucleotide sequence is SEQ ID NO.

1.

2. The protein BpfA encoded by the oxidase gene bpfA according to claim 1, characterized in that The amino acid sequence is SEQ ID NO.

2.

3. A recombinant expression vector containing the oxidase gene bpfA according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that The oxidase gene bpfA according to claim 1 is inserted between the NdeI and XhoI sites of pET-29a(+).

5. A genetically engineered bacterium containing the oxidase gene bpfA according to claim 1.

6. The genetically engineered bacterium according to claim 5, characterized in that The genetically engineered bacteria uses Escherichiacoli BL21 (DE3) as the starting strain.

7. Use of the oxidase gene bpfA according to claim 1 in the degradation of bisphenol F.

8. Use of the recombinant expression vector containing the oxidase gene bpfA according to claim 3 or 4 in the degradation of bisphenol F.

9. Use of the oxidase protein BpfA according to claim 2 in the degradation of bisphenol F.

10. Use of the oxidase protein BpfA according to claim 2 in preparing a reagent for removing bisphenol F from water or soil or in removing bisphenol F from water or soil.

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