Biomineralization surface display bacterial laccase filter and preparation and application methods thereof

Abstract

The invention discloses a biomineralization surface display bacterial laccase filter and a preparation and application method thereof, and relates to the technical field of phenol pollutant detection, the biomineralization surface display bacterial laccase filter comprises a needle type filter, a layer of mineralized laccase cells is fixed on a filter membrane of the needle type filter, and a mineralized laccase cell layer is formed; the mineralized laccase cells are prepared by selecting escherichia coli with bacterial laccase displayed on the surface and performing biological mineralization by using a phosphate buffer solution containing metal ions; the biomineralization surface display bacterial laccase filter membrane is applied to detection of phenol, m-aminophenol and p-nitrophenol and quantitative analysis of the three substances. The bacterial laccase can directly react on the cell surface after being subjected to a surface display technology, the activity and stability of the laccase are further improved after biomineralization, and the method has the advantages of short operation time, convenience, interference resistance, sensitivity and visual detection.

Description

Technical Field

[0001] The invention relates to the technical field of phenol pollutant detection, in particular to a biomineralized surface-displaying bacterial laccase filter and a preparation and application method thereof. Background Art

[0002] Existing methods for detecting phenolic compounds include spectrophotometry, gas chromatography, liquid chromatography, and constant potentiostatic titration. Among them, spectrophotometry and liquid chromatography require more equipment and reagents such as pure water and standards, and are greatly affected by other interference factors; gas chromatography requires a longer separation time and higher equipment cost; constant potentiostatic titration requires higher equipment cost and professional technology; compared with these methods, colorimetric analysis has the advantages of simplicity, visualization and rapid analysis, so it is often used in real-time environmental monitoring and industrial processes.

[0003] However, in the traditional colorimetric method, 4-aminoantipyrine can react with phenolic compounds under alkaline conditions to generate red dyes. Potassium ferrocyanide, as an oxidant, can accelerate this reaction. The color depth of the dyes generated by different phenolic compounds is different. For example, phenol is the simplest phenolic compound, containing a hydroxyl group directly connected to the benzene ring. Its electron donation ability is relatively weak because the hydroxyl group is an electron-donating group, but its position effect is the smallest on the benzene ring. When phenol reacts with 4-aminoantipyrine, the color of the dye generated may be relatively light. m-Aminophenol contains an amino group at the meta position on the benzene ring, which is a strong electron-donating group. Due to the electron-donating effect of the amino group, it can increase the electron density on the benzene ring, so that the color of the dye generated by the reaction with 4-aminoantipyrine may be darker than phenol. p-Nitrophenol contains a nitro group at the para position on the benzene ring, which is an electron-withdrawing group. The presence of the nitro group reduces the electron density on the benzene ring, which may reduce the color depth of the dye generated by the reaction with 4-aminoantipyrine. However, the traditional colorimetric method is not very effective in distinguishing phenol, m-aminophenol and p-nitrophenol. In addition, the color, turbidity and reduced sulfide of the water sample will affect the detection quality of phenol, so additional pretreatment is required.

[0004] The application of bacterial laccase in phenolic compound detection technology has improved the ability to distinguish between different phenolic compounds, but the activity of the enzyme usually decreases after surface display, and the existing technology of using microbial cells as immobilized substrates is unstable. The stability of these surface-displayed enzymes needs to be strengthened.

[0005] Based on this, the present invention aims to provide a biomineralized surface-displayed bacterial laccase filter and a preparation and application method thereof, so as to solve the problem that bacterial laccase has low stability and reduced activity during the detection of phenolic compounds. Summary of the invention

[0006] In order to make up for the shortcomings of the existing technical problems, the purpose of the present invention is to provide a biomineralized surface-displayed bacterial laccase filter and its preparation and application methods, and to improve the activity and stability of the laccase by biomineralizing the bacterial laccase; in addition, the present invention fixes the mineralized laccase cells on an ordinary filter membrane, and pretreatment is carried out simultaneously during the detection process, making the detection fast, convenient and easier to operate.

[0007] To achieve the above object, the present invention provides the following technical solutions: A biomineralized surface-displaying bacterial laccase filter comprises a syringe filter, on the filter membrane of which a layer of mineralized laccase cells is fixed to form a mineralized laccase cell layer; the mineralized laccase cells are prepared by biomineralization using Escherichia coli displaying bacterial laccase on the surface using a phosphate buffer containing metal ions.

[0008] In the present invention, the thickness of the mineralized laccase cell layer is 1-50 μm.

[0009] In the present invention, the method for preparing the mineralized laccase cells comprises: (1) inoculating and culturing Escherichia coli carrying a surface-displayed bacterial laccase gene, and after culturing, adding isopropyl β-D-thiogalactosidase to induce bacterial laccase expression, thereby obtaining an Escherichia coli solution displaying bacterial laccase on the surface; (2) centrifuging and collecting the Escherichia coli solution displaying bacterial laccase, washing it with phosphate buffer or acetic acid-sodium acetate buffer, suspending the washed Escherichia coli displaying bacterial laccase in phosphate buffer, centrifuging it again, washing it with phosphate buffer, repeating this process several times, and finally resuspending it with phosphate buffer; (3) Adding an aqueous solution of copper sulfate, aluminum sulfate, calcium chloride or chromium chloride to the PBS solution of Escherichia coli containing surface-displayed bacterial laccase in step (2), and collecting the mineralized laccase cells by centrifugation and washing with PBS buffer until a colored precipitate is clearly visible.

[0010] In the above step (3), 15-25 μL of a 0.5-5 mM aqueous solution of copper sulfate, aluminum sulfate, calcium chloride or chromium chloride is added to 1500-2500 μL of PBS buffer containing Escherichia coli with surface-displayed bacterial laccase at 2-8°C.

[0011] A method for preparing a biomineralized surface-displayed bacterial laccase filter, comprising: (1) Aspirate the PBS buffer containing mineralized laccase cells into a syringe; (2) Install a syringe filter at the lower end of the syringe and slowly push the mineralized laccase cell solution out of the syringe so that the mineralized laccase cells adhere to the filter membrane of the syringe filter to form a mineralized laccase cell layer on the filter membrane, which is then air-dried at room temperature.

[0012] The OD of the mineralized laccase cells described in step (1) above 600 Value 0.1-0.5.

[0013] The invention discloses an application method of a biomineralized surface-displayed bacterial laccase filter. The biomineralized surface-displayed bacterial laccase filter is used to detect phenol, m-aminophenol, p-nitrophenol, lignin, hydroquinone, pyrogallol, o-chlorophenol and o-aminophenol, and to perform quantitative analysis on these substances.

[0014] A method for applying a biomineralized surface-displaying bacterial laccase filter comprises the steps of sucking a Tris-HCl buffer solution containing target phenol and 4-aminoantipyrine into a syringe, injecting the solution into a filter membrane, and then contacting the solution with a filter membrane containing M-CSD-BLac for a period of time, then injecting a reaction filtrate, and performing colorimetric identification on the reaction filtrate.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The biomineralized surface-displayed bacterial laccase filter of the present invention forms a mineralized laccase cell layer by fixing a layer of mineralized laccase cells on a filter membrane; the mineralized laccase cells are prepared by biomineralization using Escherichia coli displaying bacterial laccase on the surface and using a PBS buffer containing metal ions; the bacterial laccase can directly react on the cell surface after the surface display technology, and the activity and stability of the laccase are further improved after biomineralization; the high activity of the mineralized laccase can accelerate the detection speed; the high stability enables the biomineralized surface-displayed bacterial laccase filter membrane to be reused, thereby reducing the detection cost.

[0016] 2. The porous structure of the mineralized laccase cell layer of the present invention and the structure of the filter holes of the filter membrane in which part of the M-CSD-BLac is inserted make the filtration of smaller colored impurities in real water samples more powerful. Therefore, during the detection process, pretreatment work is carried out simultaneously, which can reduce the interference problem caused by impurities in the actual sample.

[0017] 3. The present invention, by virtue of the high activity catalysis of M-CSD-BLac, provides the advantages of short operation time (10 minutes), convenience, anti-interference, sensitivity and visualization for detection. It can be used for on-site real-time colorimetric detection of phenolic substances in water samples and has high commercial value.

[0018] 4. The biomineralized surface-displayed bacterial laccase filter membrane of the present invention can sensitively detect phenol, m-aminophenol and p-nitrophenol, and effectively distinguish phenol, m-aminophenol, p-nitrophenol, lignin, hydroquinone, pyrogallol, o-chlorophenol and o-aminophenol in real water samples, and perform quantitative analysis on these substances; it has a strong ability to distinguish between different phenolic compounds and is worthy of promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of the preparation and application of the biomineralized surface display bacterial laccase filter.

[0020] Figure 2 The figure is a comparison of the activities of mineralized laccase cells obtained by mineralization of different phosphates immobilized on the membrane of the present invention.

[0021] Figure 3 The figure is a three-dimensional porous structure formed by the mineralized laccase cells on the filter membrane surface of the present invention.

[0022] Figure 4 This is a cross-sectional view of the mineralized laccase cell layer of the present invention.

[0023] Figure 5 This is a diagram of the holes into which the mineralized laccase cells of the present invention are embedded.

[0024] Figure 6 This is a comparison chart of the filtration quality between the present invention and the existing filtration technology.

[0025] Figure 7 The diagram is a schematic diagram of the quantitative detection of phenol, m-aminophenol and p-nitrophenol according to the present invention.

[0026] Figure 8 This is a statistical diagram of the present invention for distinguishing three phenolic pollutants: phenol, m-aminophenol and p-nitrophenol. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0028] like Figure 1-8 As shown, the present invention relates to a biomineralized surface-displayed bacterial laccase filter and a preparation and application method thereof, wherein the biomineralized surface-displayed bacterial laccase filter comprises a syringe filter, a layer of mineralized laccase cells is fixed on the filter membrane of the syringe filter to form a mineralized laccase cell (M-CSD-BLac) layer; the mineralized laccase cells are selected from bacterial laccase (CSD-BLac) displayed on the surface of Escherichia coli, and are prepared by biomineralization using a PBS buffer containing metal ions.

[0029] In the present invention, the thickness of the mineralized laccase cell (M-CSD-BLac) layer is 1-50 μm, preferably 20 μm.

[0030] The preparation method of the mineralized laccase cell (M-CSD-BLac) of the present invention comprises: (1) Escherichia coli carrying the expressed laccase gene was seeded with a 1% inoculum. When the optical density of E. coli at 600 nm reached 1-2, isopropyl β-D-thiogalactosidase (IPTG) was added at a final concentration of 1-2 mM and further cultured overnight at 16°C and 200 rpm to induce bacterial laccase expression. (2) The expressed bacterial solution was centrifuged at 4-5°C and 5000-6000 rpm for 5-6 minutes; after collection, the bacteria were washed with HAc-NaAc buffer at pH 6 and finally suspended in PBS to obtain an E. coli solution displaying bacterial laccase on the surface; subsequently, the produced surface-displaying bacterial laccase E. coli was collected by centrifugation at 5000-6000 rpm for 5-6 minutes, washed and resuspended in phosphate buffer (PBS, 100 mM, pH 7); (3) Add 15-25 μL of 0.5-5 mM aqueous solution of copper sulfate, aluminum sulfate, calcium chloride or chromium chloride to 1500-2500 μL of PBS buffer containing Escherichia coli displaying bacterial laccase at 2-8°C; after one day, a colored precipitate is clearly visible, and then M-CSD-BLac is collected by centrifugation at 5000-6000 rpm for 5-6 minutes and washing with PBS buffer.

[0031] In step (3) of the present invention, copper phosphate aqueous solution is preferred. 15-25 μL of the collected M-CSD-BLac solution mineralized with different phosphates is taken out and added to a HAc-NaAc buffer solution (pH 4) containing 15-25 μL of 2,2′-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt solution, respectively, and the color of the solution is observed, and the full wavelength is measured and the absorbance value at 420 nm is compared. The results show that the reaction solution of the surface-displayed bacterial laccase mineralized with copper phosphate has the darkest color and the highest activity ( Figure 2 ).

[0032] A method for preparing a biomineralized surface-displayed bacterial laccase filtration membrane, comprising: (1) Resuspend M-CSD-BLac in PBS buffer (pH 7.4). The OD of M-CSD-BLac is 600 value 0.1 and inject into the syringe; (2) Install a syringe filter at the lower end of the syringe and slowly push out the M-CSD-BLac solution so that the mineralized laccase cells adhere to the filter membrane of the syringe filter, forming an M-CSD-BLac layer on the filter membrane, which is then air-dried at room temperature.

[0033] The biomineralized surface-displayed bacterial laccase filter membrane of the present invention is used to detect phenol, m-aminophenol, p-nitrophenol, lignin, hydroquinone, pyrogallic acid, o-chlorophenol and o-aminophenol, and quantitatively analyzes these substances, as follows: A Tris-HCl buffer solution (pH 8, 50 mM) containing the target phenol and 4-aminoantipyrine was drawn into a syringe and allowed to contact with the filter membrane containing M-CSD-BLac for 10 minutes. The reaction filtrate was then drawn out and the reaction filtrate was subjected to colorimetric analysis.

[0034] The filter containing the M-CSD-BLac layer exhibited excellent filtration quality, and its morphology was observed by scanning electron microscopy (SEM). The results showed that M-CSD-BLac was evenly distributed on the surface of the filter membrane and formed a three-dimensional porous structure ( Figure 3 ). Cross-sectional SEM image ( Figure 4 ) showed that the thickness of the M-CSD-BLac layer was about 20 μm. In addition, the SEM image of the filter membrane containing low concentration of M-CSD-BLac showed that multiple M-CSD-BLac were embedded in the pores, resulting in a decrease in pore size ( Figure 5 ). Therefore, this three-dimensional layered porous structure formed on the filter improves its filtration quality compared to conventional commercial filters and simplifies the operation by removing smaller impurities, pigments and macromolecules in actual samples ( Figure 6 ).

[0035] In order to verify the feasibility of the rapid detection method of monophenolic pollutants, the present invention selected phenol, m-aminophenol, p-nitrophenol, lignin, hydroquinone, pyrogallic acid, o-chlorophenol and o-aminophenol as representative models of phenolic pollutants. By naked eye observation, the hydrolysis products of different phenolic substances showed different colors. In the presence of a multifunctional filter containing an M-CSD-BLac layer, the superoxide anion radical generated by the decomposition of oxygen captured a proton from phenol to form a quinone radical, which then reacted with 4-aminoantipyrine to form a red phenol-quinone imine ( Figure 7A), which can not only be observed by naked eyes, but also detected by spectrophotometer. In the above detection device, as the concentration of phenol increases, the reaction solution gradually turns dark red. In order to quantitatively detect phenol, the absorbance of the reaction solution was measured, and as the concentration of phenol increases, the maximum absorption peak at 500 nm gradually increases. Then, the standard working curve was drawn, and the regression equation obtained was y = 0.609x + 0.044 (R 2 = 0.993, with a linear range of 10-70 µm. Subsequently, the detection device was used to detect m-aminophenol ( Figure 7 B). As the concentration of m-aminophenol increased, the reaction solution gradually turned into a darker orange. The absorbance of the reaction solution at 450 nm was recorded for the quantitative detection of m-aminophenol. Afterwards, a standard working curve was established, and the regression equation obtained was y = 0.689x + 0.044, with a linear range of 20~80 µm and a LOD of 0.28 µm. In addition, with the gradual increase of p-nitrophenol, a distinct yellow color was observed in the reaction solution ( Figure 7 C). Similarly, a calibration curve was established by monitoring the absorbance at 390 nm, with a regression equation of y = 5.339x + 0.069 and a linear range of 15-110 µm.

[0036] By using principal component analysis (PCA), as shown in Figure I, the three phenolic compounds were clearly distinguished and classified; moreover, they were accurately detected even in the low concentration range (0.01 ̶ 0.09 mM) ( Figure 8 ).

[0037] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A biomineralized surface display bacterial laccase filter, including a syringe filter, characterized in that: A layer of mineralized laccase cells is fixed on the filter membrane of the syringe filter to form a mineralized laccase cell layer; The mineralized laccase cells are prepared by biomineralization using Escherichia coli with bacterial laccase displayed on the surface, using a phosphate buffer containing metal ions.

2. The biomineralized surface-displayed bacterial laccase filter according to claim 1, characterized in that: The thickness of the mineralized laccase cell layer is 1-50 μm.

3. The biomineralized surface-displayed bacterial laccase filter according to claim 1, characterized in that: The preparation method of the mineralized laccase cell comprises: (1) inoculating and culturing Escherichia coli carrying a surface-displayed bacterial laccase gene, and after culturing, adding isopropyl β-D-thiogalactosidase to induce bacterial laccase expression, thereby obtaining an Escherichia coli solution displaying bacterial laccase on the surface; (2) centrifuging and collecting the Escherichia coli solution displaying bacterial laccase, washing it with phosphate buffer or acetic acid-sodium acetate buffer, suspending the washed Escherichia coli displaying bacterial laccase in phosphate buffer, centrifuging it again, washing it with phosphate buffer, repeating this process several times, and finally resuspending it with phosphate buffer; (3) Adding an aqueous solution of copper sulfate, aluminum sulfate, calcium chloride or chromium chloride to the PBS solution of Escherichia coli containing surface-displayed bacterial laccase in step (2), and collecting the mineralized laccase cells by centrifugation and washing with PBS buffer until a colored precipitate is clearly visible.

4. A biomineralized surface-displayed bacterial laccase filter as claimed in claim 3, characterized in that: In step (3), 15-25 μL of a 0.5-5 mM aqueous solution of copper sulfate, aluminum sulfate, calcium chloride or chromium chloride is added to 1500-2500 μL of PBS buffer containing Escherichia coli displaying bacterial laccase at 2-8°C.

5. A method for preparing a biomineralized surface-displayed bacterial laccase filter according to any one of claims 1 to 4, characterized in that: include: (1) Aspirate the PBS buffer containing mineralized laccase cells into a syringe; (2) Install a syringe filter at the lower end of the syringe and slowly push the mineralized laccase cell solution out of the syringe so that the mineralized laccase cells adhere to the filter membrane of the syringe filter to form a mineralized laccase cell layer on the filter membrane, which is then air-dried at room temperature.

6. The method for preparing the biomineralized surface-displayed bacterial laccase filter according to claim 5, characterized in that: The OD of the mineralized laccase cells 600 Value 0.1-0.

5.

7. A method for using the biomineralized surface-displayed bacterial laccase filter according to any one of claims 1 to 4, characterized in that: The biomineralized surface-displayed bacterial laccase filter is used to detect phenol, m-aminophenol, p-nitrophenol, lignin, hydroquinone, pyrogallol, o-chlorophenol and o-aminophenol, and to perform quantitative analysis on these substances.

8. The application method of the biomineralized surface-displayed bacterial laccase filter according to claim 7, characterized in that: A Tris-HCl buffer solution containing the target phenol and 4-aminoantipyrine is sucked into a syringe, injected onto the filter membrane, and then contacted with the mineralized laccase cell layer for a period of time, and then the reaction filtrate is pumped out and the reaction filtrate is colorimetrically identified.

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