Application of Mn (II) oxidase PomA in preparation of dye decolorizing agent

Abstract

The invention discloses application of Mn (II) oxidase PomA in preparation of a dye decolorizing agent. The amino acid sequence of the Mn (II) oxidase PomA is as shown in SEQ ID NO. 2.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically relates to the application of Mn(II) oxidase PomA in the preparation of a dye decolorizing agent. Background Art

[0002] With the rapid development of industrialization and the acceleration of urbanization, the organic and inorganic pollutants enriched in wastewater pose a serious threat to the environment and human health. One of the common wastewater pollutants is heavy metals. Due to their non-biodegradability and long half-lives, they easily cause pollution of groundwater, surface water, soil, and crops. Another common wastewater pollutant is dyes. Because of their relatively stable molecular structures, they are also difficult to be biodegraded and also pose a great threat to the ecosystem. Therefore, it is of great significance to propose a low-cost and efficient treatment technology for Mn(II) and dye wastewater.

[0003] In recent years, wastewater treatment technologies based on microbial enzymes have attracted much attention. Microbial enzymes are a class of catalytically active biological macromolecules produced by microorganisms, which can catalyze the transformation and degradation of organic and inorganic substances in wastewater, significantly reducing the toxicity and pollution level of wastewater. In terms of heavy metal decontamination, microbial enzymes can convert soluble ionic heavy metals into solid heavy metal oxides, or convert heavy metal ions with high-toxicity valence states into heavy metal ions with low-toxicity valence states. In terms of dye decontamination, microbial enzymes can accelerate redox reactions, break the unsaturated bonds or chromogenic groups of dye molecules, and convert complex organic structures into simple inorganic substances. In practical applications, wastewater treated with microbial enzymes generally has the advantages of recoverability of target substances, no secondary pollution, high efficiency and economy, and strong universality. This treatment method is conducive to the sustainable development of environmental protection. The enzymes mainly used for enzymatic dye decolorization are lignin-degrading enzymes, such as lignin peroxidase and laccase, but their insufficient stability, low decolorization efficiency and high large-scale production costs limit their wide application. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provides the application of Mn(II) oxidase PomA in the preparation of a dye decolorizing agent.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows: The Mn(II) oxidase PomA used in the present invention for the preparation of a dye decolorizing agent is derived from the Mn(II) oxidizing bacterium Providencia manganoxydans LLDRA6 (preserved in the China Center for Type Culture Collection, with the strain number CCTCC AB 2021154T; CCTCC NO: M2018876). First, the total DNA of strain LLDRA6 is extracted, and primers are designed to clone the target gene pomA(GenBank accession: PQ362382) was heterologously expressed using the Escherichia coli expression strain BL21 and the expression vector pET32a(+), and a recombinant vector and a recombinant bacterium were obtained. The crude enzyme solution was obtained by ultrasonic disruption, and the Mn(II) oxidase PomA that could be used for dye decolorization was separated and purified using Ni-NTA agarose protein purification resin.

[0006] The amino acid sequence of the Mn(II) oxidase PomA is shown in SEQ ID NO.2. The coding nucleotide sequence of the Mn(II) oxidase PomA is shown in SEQ ID NO.1.

[0007] The recombinant vector contains the nucleotide sequence shown in SEQ ID NO.1.

[0008] The recombinant bacterium contains the aforementioned recombinant vector.

[0009] The present invention also provides an enzyme solution of a dye decolorizing agent. The enzyme solution contains Mn(II) oxidase PomA. The working concentration of PomA in the enzyme solution is 0.086 mg / mL, and the working pH of the enzyme solution is 3.0 - 6.0.

[0010] The present invention shows that the Mn(II) oxidase PomA has strong oxidation ability and has broad application prospects in the field of dye decolorization and sewage treatment. Description of the Drawings

[0011] Figure 1 : PCR amplification electrophoresis map of the Mn(II) oxidase gene in the example. The electrophoresis band is clear and the size is 1632 bp; Figure 2 : Double digestion verification map of the recombinant vector pET- pomA in the example; Figure 3 : SDS-PAGE map of heterologous expression and affinity chromatography purification in the example. Among them, lane 1: induced total protein; lanes 2 - 3: miscellaneous proteins eluted with 100 mmol / L imidazole; lanes 4 - 7: target bands eluted with 125 mmol / L imidazole; Figure 4 : Verification of the Mn(II) oxidation activity of the enzyme in the example; Figure 5 : Enzymatic properties of the Mn(II) oxidation activity of the enzyme in the example; Figure 6 : Verification of the dye decolorization of the enzyme in the example. Among them, A is Congo red dye decolorization (1 is the control group, 2 - 4 are the experimental group triplicates), and B is crystal violet dye decolorization (1 is the control group, 2 - 4 are the experimental group triplicates). Detailed implementation mode

[0012] The gene of the Mn(II) oxidase is derived from Mn(II)-oxidizing bacteria Providencia manganoxydans LLDRA6 (deposited in the China Center for Type Culture Collection, strain number CCTCC AB 2021154T; CCTCC NO: M2018876). Total DNA was extracted using a bacterial genomic DNA extraction kit purchased from Sangon Biotech (Shanghai) Co., Ltd. Primers were designed to perform PCR amplification on the target gene. The nucleotide sequence of the Mn(II) oxidase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2

[0013] The specific method for obtaining the target gene is as follows: Total DNA was extracted using a bacterial genomic DNA extraction kit purchased from Sangon Biotech (Shanghai) Co., Ltd. Primers were designed to perform PCR amplification on the target gene. The amplification primers are shown in Table 1: Table 1 Primer sequences of the target gene Primer names Primer sequences (5'-3') Enzymatic cleavage sites Forward primer <![CDATA GGATCC ATGGCGGAAAATTTCCCAG (SEQ ID NO.3)]]> H I Reverse primer <![CDATA AAGCTT ACTGACCGTAAAGCCC (SEQ ID NO.4)]]> d III The PCR amplification program was pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 2 min, 32 cycles, and extension at 72°C for 10 min. The target gene was obtained pomA , see the PCR amplification electrophoresis diagram in Figure 1 , Figure 1 , and the electrophoresis band was clear and single, with a size of 1632 bp

[0014] The above PCR product was subjected to TA cloning with the T-vector pLB and transformed into Escherichia coli DH5α competent cells. Recombinant cloning plasmid pLB- was extracted pomA , and the cloned plasmid pLB- pomA and the expression vector pET-32a(+) were double-digested with Bam H I and Hin d III. The small fragment in the cloned plasmid pLB- in the double-digested product and the large fragment of the expression vector pET-32a(+) were recovered, ligated with T4 ligase, and the ligation product was transformed into Escherichia coli DH5α competent cells. After screening with Ampicillin resistance, transformants were selected and colony PCR was performed using the universal primers T7 and T7-ter. The plasmid of the positive transformant was extracted and subjected to double-digestion verification and sequencing. The results of the digestion verification are shown in pomA , and there was a bright band between 1500 - 2000 bp, indicating the target gene Figure 2 , pomASuccessfully connected to the expression vector pET-32a(+). Finally, the recombinant plasmid was sequenced, and the results showed that the target gene sequence information was correct, and the recombinant vector pET- pomA . Submitted the pomA gene sequence to NCBI, and the accession number was GenBank accession: PQ362382.

[0015] Placed 50 μL of Escherichia coli BL21(DE3) competent cells on ice to melt, took 5 μL of the above recombinant vector (pET32a(+)- pomA ) and added it to the competent cells, gently pipetted and mixed evenly, and placed it on ice for 30 min. Heat shock at 42 °C in a water bath for 90 s, take it out and place it on ice for 3 min. Add 400 μL of LB liquid medium, incubate at 37 °C in a shaker at 180 rpm for 1 h. Take 200 uL of competent cells and spread them on an LB solid plate containing Ampicillin antibiotic (final concentration 50 μg / mL), culture overnight at 37 °C, pick the transformants, and use the universal primers T7 and T7-ter for colony PCR verification. The positive transformants can be inoculated into an LB liquid medium without antibiotics and cultured overnight at 37 °C at 180 rpm. The obtained bacterial liquid is the recombinant bacterium BL21-pET- pomA .

[0016] Inoculated the Escherichia coli recombinant bacterium BL21-pET- pomA into an LB liquid medium, cultured at 37 °C in a shaker at 170 rpm until the OD600 was about 0.6, then added 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the large expression of Mn(II) oxidase. After adding IPTG, place it at 16 °C and continue to culture at 150 rpm for about 16 h. Centrifuge at 4 °C, 10000 rpm for 10 min to collect the bacterial cells, resuspend them in PBS buffer (pH = 7), and perform ultrasonic treatment with a cell disruptor (turn on for 2 s, stop for 3 s, ultrasonic for 10 min) to obtain a cell lysate and centrifuge at 4 °C, 10000 rpm for 20 min to remove cell debris. The obtained supernatant is the crude enzyme solution containing a large amount of target protein.

[0017] Purify the protein using a Ni-NTA agarose purification resin kit. Filter the above crude enzyme solution with a 0.4 μm filter head for standby. The specific purification steps are as follows: ① Allow the storage buffer in the Ni-NTA resin to flow out by gravity, balance the Ni-NTA resin with ultrapure water for 5 to 6 column volumes, and then balance it with binding buffer (PBS buffer containing 20 mM imidazole) for 5 to 6 column volumes. Use a flow rate of 0.5 to 1 mL / min to allow the buffer to slowly drain from the resin.

[0018] ② Mix the filtered crude enzyme solution with the binding buffer at a ratio of 1:1 to prepare the sample solution, so that the total volume of the sample solution is twice the volume of the column. Add the sample solution to the column and collect the flow-through into a centrifuge tube. If there is excess sample, you can re-load it. Re-circulating it once can improve the binding force between the sample and the filler.

[0019] ③ Wash the column with two column volumes of binding buffer (PBS buffer containing 20 mM imidazole) and elution buffer (PBS buffer containing 100 mM imidazole) and collect the flow-through, and test the absorbance of the flow-through at 280 nm. Repeat the steps with a new collection tube each time the buffer is changed until the absorbance of the flow-through at 280 nm is close to the baseline, and the impurities are fully removed.

[0020] ④ Elute the target protein with the target protein elution buffer (PBS buffer containing 125 mM imidazole) until the absorbance of the eluate at 280 nm is close to the baseline.

[0021] ⑤ Post-treatment of column material: elute the column material with 5 volumes of elution buffer (PBS buffer containing 200 mM imidazole), then balance the column material with 5 volumes of binding buffer (PBS buffer containing 20 mM imidazole), and finally wash the column material with 5 volumes of deionized water, add 20% ethanol protective solution, and store it at 2~8℃.

[0022] ⑥ The eluted protein can be used for SDS-PAGE analysis. Prepare SDS-PAGE electrophoresis samples and detect the purity of the target protein. The results are as follows: Figure 3 As shown. Lane 1 is the induced total protein, lanes 2-3 are the impurity protein bands eluted with 100 mmol / L imidazole, and lanes 4-7 are the target bands eluted with 125 mmol / L imidazole. As can be seen from the figure, the target band containing PomA protein is clear, with a molecular weight of about 74 kDa. Excluding the histidine tag protein (2.4 kDa) and the solubilizing tag protein (12 kDa) of pET-32a(+), the remaining part is consistent with the expected size of PomA itself, 59.4 kDa.

[0023] ⑦Dialyze and desalt the eluate containing the target protein from the previous step. The dialysis bag needs to be pretreated before use. The pretreatment steps are as follows: Cut the dialysis bag into small sections of 10 - 20 cm. Put the dialysis bag into the dialysis bag treatment solution and boil for 10 min. Thoroughly wash the inner and outer sides of the dialysis bag with distilled water. Put the washed dialysis bag into 1 mM EDTA-2Na and continue to boil for 10 min. After the dialysis bag cools down, soak it in 30% ethanol solution and store it in a 4℃ refrigerator. Take the pretreated dialysis bag, wash it clean with distilled water before use. First, clamp one end with a dialysis clip, and add the eluate containing the target protein from the other end. After adding the sample, clamp it with another dialysis clip in the same way. Note that there should be some space reserved inside the dialysis bag at this time to prevent it from bursting during dialysis. Put the dialysis bag into the pre-cooled dialysis solution, and gently stir it magnetically in an ice bath for 20 min, then put it into a 4℃ refrigerator. Change the solution every 2 h and dialyze 3 times.

[0024] ⑧Put the dialyzed sample and the dialysis bag together in a box, and use pre-cooled PEG20000 for solid embedding. After observing that PEG20000 absorbs water and becomes a paste, replace it and add new PEG20000. Observe the sample every 2 min during this period, and stop the embedding immediately when the sample becomes turbid. Then wash the PEG20000 on the surface of the dialysis bag with the dialysis solution, and aliquot the protein sample in the dialysis bag. Add 5% - 10% glycerol and store it in a -80℃ refrigerator. The concentration of the protein stock solution is determined to be 0.982 mg / mL by Bradford colorimetry.

[0025] Leucoberbelin blue (LBB) is a synthetic triphenyl complex that can reduce the high-valent Mn(III) and Mn(IV), while being oxidized itself. The reduced LBB is colorless, and the oxidized LBB is blue and shows a maximum absorption peak at 620 nm, which is widely used for the qualitative and quantitative detection of manganese oxides.

[0026] (1)Detection of Mn(II) oxidase activity and determination of the optimal reaction conditions Set the reaction system to 500 μL, containing 200 μL of the target protein PomA, 10 mmol / L HEPES buffer (pH 7.0), and 10 mmol / L MnCl 2 solution. Use ddH 2Replace the target protein PomA with O. After incubating the two reaction systems at 37 °C for 24 h, take 50 μL of each reaction solution and mix it with 250 μL of 0.04% LBB staining solution, and react in the dark for 20 min. After centrifugation, take 200 μL of the supernatant and place it in a 96-well plate, and measure its absorbance at 620 nm with an enzyme-linked immunosorbent assay (FlexA-200, Aosheng, Hangzhou). Calculate the manganese oxide content according to the KMnO 4 standard curve. According to the above reaction system, investigate the ability of PomA to oxidize Mn(II) under different pH values (7.0 - 8.2), temperatures (20 - 90 °C), and Cu(II) concentrations (0.1 - 20 mmol / L), and determine the optimal reaction conditions. Taking the highest manganese oxide production as 100%, calculate the relative activity percentage of PomA oxidizing Mn(II) under different reaction conditions. All treatments are repeated 3 times.

[0027] The test results are as Figure 4 shown. After fully reacting the blank control group solution with Mn(II) and then incubating it with LBB staining, there is basically no color change in the reaction system, showing colorless and transparent ( Figure 4 A). In contrast, after the PomA protein solution fully reacts with Mn(II) and then is stained with LBB, it turns blue ( Figure 4 B), indicating that manganese oxide is generated in the reaction system. Investigate the effects of different Cu(II) concentrations, temperatures, and pH conditions on the oxidation of Mn(II) by PomA to determine the optimal reaction conditions. As Figure 5 (A) shows, in the range of 0 - 20 mmol / L Cu(II), a lower concentration of Cu(II) (<5 mmol / L) has a stronger promoting effect on the Mn(II) oxidation ability of PomA than a higher concentration (>5 mmol / L). When the exogenous Cu(II) concentration is 5 mmol / L, the relative activity of PomA for Mn(II) oxidation reaches the highest value of 100%.

[0028] The effect of different temperatures on the Mn(II) oxidation activity of PomA is as Figure 5 (B) shows. When the temperature is below 28 °C, the relative activity of PomA for Mn(II) oxidation is relatively low, indicating that only a very small amount of Mn(II) is oxidized. When the temperature is between 37 °C and 60 °C, the relative activity of Mn(II) oxidation gradually increases and reaches 100% at 60 °C. Although the relative activity of PomA for Mn(II) oxidation begins to decline as the temperature continues to rise (>60 °C), there is still a relative activity of 60.44% at 70 °C, indicating that PomA has strong tolerance to high temperatures.

[0029] The effect of different pH values on the Mn(II) oxidation activity of PomA is as Figure 5As shown in (C), in the range of pH 6.5 - 9.0, starting from pH > 7, the relative Mn(II) oxidation activity of PomA continuously increases. When pH = 9.0, its relative activity reaches the highest value of 100%. However, it should be noted that when pH > 7.8, the relative Mn(II) oxidation activity of the blank control solution also starts to increase sharply from zero and also reaches the highest value at pH = 9.0. This phenomenon indicates that under the condition of pH > 7.8, natural oxidation of Mn(II) may occur.

[0030] (2)Determination of enzyme kinetic parameters With the optimal reaction conditions as a reference, the reaction system was set to 200 μL, and 0.982 mg / mL of purified PomA protein (final concentration E t was 1.65×10 -6 mol / L) was added, and different Mn(II) concentrations (0 - 50 mM) were set in the reaction system. After reacting for 1 h, 50 μL of the reaction solution was mixed with 250 μL of LBB staining solution and reacted in the dark for 20 min. After centrifugation, 200 μL of the supernatant was added to a 96-well plate, and the absorbance value was recorded by an enzyme-labeling instrument. OD 620 Using GraphPad Prism5 software for non-linear regression fitting according to the Michaelis-Menten equation, K m and V max other enzyme parameters were obtained. k cat From the formula k cat = V max / E t it was calculated.

[0031] Taking the optimal reaction conditions in Figure 5 as a reference, but considering the natural oxidation phenomenon of Mn(II) when pH > 7.8, Cu(II) = 5 mmol / L, T = 60°C, and pH = 7.8 were selected as the conditions for this enzyme kinetic test. Using Mn(II) as the substrate (0 - 50 mM), the enzyme kinetic constants of PomA were tested. Using GraphPad Prism5 software for non-linear regression fitting, as shown in Figure 5 (D), K m = 3.11 ± 0.02 mmol / L, V max = 3.931×10 -6 mol / (L·min), k cat = 2.38 min-1 。

[0032] The decolorization experiments of two dyes, Congo red and crystal violet, were carried out using the above-mentioned Mn(II) oxidase. Their maximum absorption peaks were at 490 nm and 590 nm respectively. The decolorization reaction system was set as 5 ml: 1 mL dye (final concentration 100 mg / L), 0.5 mL enzyme solution (final concentration 0.086 mg / mL), 0.5 mL medium ABTS (final concentration 0.5 mmol / L), and 3 mL citrate-phosphate buffer (pH = 3.0 - 6.0). The system reacted at 60 °C for 2 h. The absorbance was measured at the wavelength of the maximum absorption peak of the dye, and the absorbance was A 1 ; The same method was used to add an equal amount of inactivated enzyme solution as a control in the reaction system, and its absorbance was measured as A 0 , the dye decolorization rate (%) = (A 0 –A 1 ) / A 0 。

[0033] As Figure 6 (A) shows, when the enzyme solution reacted with Congo red dye, after 2 hours of treatment time, the observed decolorization effect was extremely significant. Through calculation, the highest dye decolorization rate reached 86.5%. This result indicates that the enzyme demonstrated high decolorization ability when treating Congo red dye. As Figure 6 (B) shows, after the enzyme solution reacted with crystal violet dye for 2 hours, an obvious decolorization effect was also achieved, and its decolorization rate reached up to 63.8% at most. This data further confirmed the effectiveness and applicability of the enzyme in the decolorization treatment of different dyes.

[0034] Based on the above experimental results, it can be concluded that this enzyme has significant ability and application value in dye decolorization. Given that it can achieve high decolorization rates when treating two different types of dyes, Congo red and crystal violet, it indicates that the enzyme may have good decolorization effects on a variety of dyes. Therefore, applying this enzyme to the field of sewage treatment for dye decolorization not only has the potential to effectively reduce the content of dyes in wastewater and reduce environmental pollution, but may also provide a new and efficient biological treatment method for the sewage treatment industry.

Claims

1. Application of Mn(II) oxidase PomA in the preparation of dye decolorizing agent, characterized in that: The amino acid sequence of the Mn(II) oxidase PomA is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The nucleotide sequence of the Mn(II) oxidase PomA is shown in SEQ ID NO.

1.

3. A recombinant vector for preparing a dye decolorizing agent, characterized in that: The recombinant vector contains the nucleotide sequence shown in SEQ ID NO.

1.

4. A recombinant bacterium for preparing a dye decolorant, characterized in that: The recombinant bacteria comprises the recombinant vector according to claim 3.

5. An enzyme solution of a dye decolorizing agent, characterized in that The enzyme solution contains Mn(II) oxidase PomA, the working concentration of PomA in the enzyme solution is 0.086 mg / mL, and the working pH value of the enzyme solution is 3.0-6.0.

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