Enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and application

[0023] The extraction of embodiment 1Pseudomonas sp.ONBA-17 total DNA (Genomic DNA)

[0024] 1.1 Expanded cultivation of Pseudomonas sp.ONBA-17

[0025] Under a sterile operating environment, small pieces of Pseudomonas sp.ONBA-17 stored at -70°C were picked with an inoculation needle, placed and spread on Luria-Bertani (LB) solid medium. 28 ℃, static culture 3d. A single colony was selected, and after two transfers, it was observed that the colonies on the plate had the same shape (no bacteria). The "plate" and Luria-Bertani (LB) solid/liquid medium described here are all common terms, medium, techniques and methods in the field of microbial research/production. For details, see "Molecular Cloning Experiment Guide" (J. Sam Brook, et al. Molecular Cloning Experiment Guide (Third Edition) [M]. Huang Peitang, et al. Translated. Beijing: Science Press, 2008). Unless otherwise specified, the techniques, methods, culture media, reagents and medicines used in microbial research are all commonly used in microbiological research, and are all recorded in the "Molecular Cloning Experiment Guide".

[0026] 1.2 Extraction of total DNA of Pseudomonas sp.ONBA-17

[0027] Total bacterial DNA was extracted by SDS high-salt precipitation method. Pick a single colony on the LB plate, connect to 100mL LB liquid medium and cultivate to the stationary phase. Collect the bacteria by centrifugation, add an equal volume of TEN solution [10mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0), 0.1M NaCl] to wash the bacteria, collect the bacteria by centrifugation, and suspend in 10mL TE [10mM Tris -Cl (pH8.0), 1mM EDTA (pH8.0)] solution, add 100μL lysozyme (100mg/mL), 37℃ water bath for 1h, add 40μL proteinase K (20mg/mL), then add 300μL 20% (w /v%) SDS, 37 ° C water bath overnight (about 12h). Add 1/2 volume of saturated NaCl and shake vigorously, centrifuge at 12000g for 10min, extract the supernatant twice with an equal volume of phenol: chloroform, centrifuge at 12000g for 10min, collect the supernatant, add an equal volume of TE solution (dilution to adjust NaCl concentration), 0.6 times the volume Precipitate with isopropanol, centrifuge at 12000 g for 15 min, wash the precipitate with 70% (v/v%) ethanol, and dissolve in 100 μL TE after ethanol evaporates.

[0028] After appropriate dilution of the total DNA of the extracted thalline, its quality was detected by 0.75% (w/v%) agarose electrophoresis (such as figure 1 As shown), it was found that most of the extracted total DNA was concentrated at about 23kb, basically no RNA existed, the purity was better, and the concentration was higher.

[0029] The acquisition of embodiment 22-NBA degrading enzyme coding gene nbaB

[0030] After a large number of analysis experiments, a pair of effective amplification primers (denoted as SEQ ID No.3 and SEQ ID No.4 respectively) were screened out from 24 pairs of self-designed primers.

[0031] nbaB-f,5'-ACGAC CATATG AGTTACCAAAACTTAG-3' (the underline is the NdeI restriction site, and the gene sequence is SEQ ID No.3);

[0032] nbaB-r, 5'-CATCA GAATTC GGAAGACCAGGAGC-3' (the underline is the EcoRI restriction site, and the gene sequence is SEQ ID No.4).

[0033] 25 μL amplification system: 2.5 μL of 10×Taq DNA polymerase reaction buffer; 2 μL of dNTP (25 mM); 0.5 μL of each primer (25 pmol/μL); Mg 2+ Buffer (25mM) 2.5μL; total DNA obtained in Example 1 0.5μL (about 100ng); Taq enzyme (5U/μL) 0.3μL; ddH 2 016.2 μL. Reaction parameters: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 1 min, amplification for 25 cycles, and final extension at 72°C for 5 min.

[0034] The amplified product was detected by 0.75% (w/v%) agarose electrophoresis, and it was found that the amplified fragment was consistent with the expected size (about 1.9kb). Cut out the gel containing the nbaB gene amplification fragment, put it into a sterile 1.5mL centrifuge tube and weigh it, use the BIOMIGA Gel Recovery and Purification Kit to recover the target fragment, and store it at 4°C for future use.

[0035] The obtained PCR products were subjected to TA cloning and subsequent operations according to the "Molecular Cloning Experiment Guide". The vector pMD19-T for cloning and related reagents were purchased from TaKaRa Biotechnology Co., Ltd. (TaKaRa, Dalian). After picking the plasmid and verifying the correct size of the inserted fragment by agarose gel electrophoresis, pick the clone and submit it to Shanghai Sangon for sequencing. The obtained nbaB coding gene sequence is shown in SEQ ID No.2, and its corresponding amino acid sequence is shown in SEQ ID No.1. The gene sequence comparison was first logged on the website of the National Center for Biotechnology Information (NCBI), and then searched GenBank through the BLAST search engine.

[0036] The results showed that the highest sequence homology of the functional gene homologous to the nbaB coding gene sequence was only 84% (U49504.1), and there was no report about the degradation of 2-NBA in the gene referred to by U49504.1. However, the highest sequence homology of the protein homologous to the amino acid sequence of NbaB is only 82% (YP_987297.1), and the dioxygenase referred to by YP_987297.1 has not been reported on the degradation of 2-NBA.

[0037] Construction, expression and purification of embodiment 3 expression vector

[0038] The amplified product of the nbaB gene in Example 2 was digested with Nde I and EcoRI, and then the fragment was inserted into the pET21b(+) vector digested with the same restriction endonuclease. In the recombinant plasmid, the nbaB gene is driven by the T7 promoter, the C-terminus of the expression product has 6 histidine tags, and ampicillin is the selection marker. The joined fragments were verified by Shanghai Sangon sequencing to ensure that no mutations were introduced during the PCR process.

[0039] The recombinant plasmid was transferred into Escherichia coli BL21 (DE3), and then the strain containing the recombinant plasmid (numbered Y-6) was selected, and the foreign gene introduction was verified by Shanghai Sangon sequencing. Y-6 was cultured with LB based on 37°C culture, after A 600nm When the value was 0.6, IPTG was added to a final concentration of 1 mM, followed by overnight expression at 20° C. to avoid inclusion bodies. Cells were collected by centrifugation, resuspended in 50 mM sodium phosphate buffer (pH 7.5), and disrupted by ultrasonication (power 400 W) in an ice-water bath—sonication for 5 seconds, pause for 15 seconds, and a total of 60 cycles. Subsequently, cell debris was removed by centrifugation at 10,000 g for 30 min at 4°C. The supernatant is the crude enzyme extract and stored at low temperature. At the same time as the experiment in this step, E.coli BL21 (DE3) which has not been transformed into a recombinant plasmid was set as a control, and the steps of induction, centrifugation, crushing and collection of crude enzyme extract were also carried out.

[0040] The above-mentioned crude enzyme extract was purified using a one-stop histidine-tagged protein purification kit (Ni-NTA Spin Kit, QIAGEN) to obtain a purified enzyme liquid sample (the pure enzyme content of o-nitrobenzoic acid degrading enzyme was 2.5 mg/mL), all operations were carried out under low temperature environment.