Rapid quintozene detection method based on enzyme catalytic reaction

Abstract

The invention belongs to the technical field of biology, and particularly relates to a pentachloronitrobenzene rapid detection method based on enzyme catalysis reaction, which comprises the following steps: converting pentachloronitrobenzene into pentachlorophenol and nitrate radicals through an enzyme system consisting of iron thioflavin protein reductase, ferredoxin and nitrobenzene dioxygenase; and then nitrate radicals are specifically detected by a Griess reagent method so as to indirectly reflect quintozene. The detection method disclosed by the invention is good in specificity, short in detection time and remarkable in visual detection effect, and can be used for on-site rapid detection of quintozene.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an enzymatic reaction system for visual detection of pentachloronitrobenzene. Background Technology

[0002] Pentachloronitrobenzene (PCNB) is a widely used organochlorine protective fungicide, primarily used to control seedling blight, damping-off, and anthracnose in vegetables. It is also a common seed dressing and soil treatment agent in the cultivation of crops such as potatoes, wheat, cotton, tomatoes, and ginseng. However, due to its chemical stability, its half-life in soil is as long as 5-10 months, and even as long as 1042 days in water. Furthermore, it is highly soluble in lipids. Therefore, its residues can damage organs such as the heart, reproductive system, and liver in mammals, and can even be fatal at certain concentrations.

[0003] Currently available pesticide detection methods mainly include gas chromatography (GC), liquid chromatography (LC), GC-MS / MS, and LC-MS / MS. Although these methods have high sensitivity and accuracy and low detection limits, the instruments are expensive, the operation is difficult, and the detection time is long, making it impossible to achieve rapid detection. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting pentachloronitrobenzene using an enzyme-catalyzed reaction and the Griess reagent method. The method involves converting pentachloronitrobenzene into pentachlorophenol and nitrite using an enzyme system, followed by color development using the Griess reagent method—a nitrite-specific detection method—to indirectly reflect the pentachloronitrobenzene reaction.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The present invention provides a composition comprising: ferroflavin protein reductase, feroxin, and nitrobenzene dioxygenase.

[0007] In some specific embodiments of the present invention, the ferrous thioflavone protein reductase of the above composition has:

[0008] (1) The amino acid sequence as shown in SEQ ID NO: 1; or

[0009] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0010] (3) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2).

[0011] In some specific embodiments of the present invention, the ferroredoxin in the above composition has:

[0012] (4) The amino acid sequence as shown in SEQ ID NO: 2; or

[0013] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0014] (6) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5).

[0015] In some specific embodiments of the present invention, the α subunit of the nitrobenzene dioxygenase in the above composition has:

[0016] (7) The amino acid sequence as shown in SEQ ID NO: 3; or

[0017] (8) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (7), and whose function is the same as or similar to that of (7); or

[0018] (9) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (7) or (8);

[0019] The β subunit of the nitrobenzene dioxygenase has:

[0020] (10) The amino acid sequence as shown in SEQ ID NO: 4; or

[0021] (11) An amino acid sequence obtained by substitution, deletion or addition of one or more residues as shown in (10), and whose function is the same as or similar to that of (10); or

[0022] (12) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (10) or (11).

[0023] In some specific embodiments of the present invention, the plurality of the above composition is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0024] The present invention also provides the use of the above composition in the detection of pentachloronitrobenzene or in the preparation of products for detecting pentachloronitrobenzene.

[0025] The present invention also provides a detection reagent comprising the above-described composition.

[0026] In some specific embodiments of the present invention, the above-mentioned detection reagent also includes Griess reagent.

[0027] In some specific embodiments of the present invention, the Gris reagent of the above-mentioned detection reagent includes at least one of p-aminobenzenesulfonic acid or its derivatives, α-naphthylamine or its derivatives, and naphthol or its derivatives.

[0028] In some specific embodiments of the present invention, the Griess reagent of the above-mentioned detection reagent includes p-aminobenzenesulfonic acid and naphthylethylenediamine hydrochloride.

[0029] The present invention also provides an apparatus comprising the above-described composition or the above-described detection reagent.

[0030] This invention also provides a method for detecting pentachloronitrobenzene, comprising detection based on any of the following:

[0031] i) The above-mentioned composition;

[0032] ii) The above-mentioned test reagents;

[0033] iii) The above-mentioned device.

[0034] In some specific embodiments of the present invention, the above detection method includes: mixing the sample to be tested with NADH and the composition, mixing with p-aminobenzenesulfonic acid, and then mixing with naphthylethylenediamine hydrochloride to obtain the detection result.

[0035] In some specific embodiments of the present invention, the above detection method includes: mixing the sample to be tested with NADH and the composition at 32-35°C (which can be 32.5°C, 33°C, 33.5°C, 34°C or 34.5°C) and 50-150 rpm (which can be 60 rpm, 70 rpm, 80 rpm, 90 rpm, 110 rpm, 120 rpm, 130 rpm or 140 rpm) for 30-60 min (which can be 35 min, 45 min, 50 min or 55 min), reacting with p-aminobenzenesulfonic acid for more than 3 min (which can be 4 min, 6 min, 8 min or 10 min), and then reacting with naphthylethylenediamine hydrochloride for more than 10 min (which can be 11 min, 12 min, 13 min, 14 min, 20 min or 30 min) to obtain the detection result;

[0036] The molar ratio of ferrosulfonamide protein reductase, ferroredoxin, and nitrobenzene dioxygenase in the composition is (1~2):(1~2):(1~2), which can be 1:1:1, 2:1:1, 1:1:2, 2:2:1, 2:1:2, or 1:2:2.

[0037] The final concentration of NADH is 0.1~0.3 mM, which can be 0.15 mM or 0.25 mM.

[0038] In some specific embodiments of the present invention, the preparation method of the ferrosulfonase, the ferricoxane, or the nitrobenzene dioxygenase in the above detection method includes:

[0039] S1: Cultivate engineered bacteria, wherein the engineered bacteria express the iron sulfoflavin protein reductase, the ferricredoxin and / or the nitrobenzene dioxygenase, to obtain a bacterial solution;

[0040] S2: IPTG, L-cysteine, and ferrous sulfate are mixed with the bacterial culture to make the final concentrations of IPTG, L-cysteine, and ferrous sulfate 0.4 mM, 0.05 mM, and 0.1 mM, respectively. After induction, the bacterial cells are collected, the supernatant is collected after lysis to obtain crude enzyme solution. After purification, the iron thioflavin protein reductase, the ferricredoxin, and / or the nitrobenzene dioxygenase are obtained.

[0041] The advantages of this invention compared to the prior art are as follows:

[0042] This invention provides a system that can enzymatically convert pentachloronitrobenzene and produce a colorimetric result, enabling effective and rapid detection of pentachloronitrobenzene. The system is easy to operate, shortens instrument detection time, and reduces detection costs.

[0043] The detection limit of the pentachloronitrobenzene method provided by this invention is 0.0566 mg / kg. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0045] Figure 1 Electrophoresis diagram of PAGA enzyme system composition;

[0046] Figure 2 Show the nitrite standard curve;

[0047] Figure 3 The actual phenomena after testing and the blank control are shown;

[0048] Figure 4 The purple-red compounds formed at different reaction temperatures correspond to A. 540 and the nitrite concentration obtained after conversion;

[0049] Figure 5 The purple-red compound formed at different reaction times corresponds to A. 540 and the nitrite concentration obtained after conversion;

[0050] Figure 6 The purple-red compounds generated at different buffer pH values ​​correspond to A. 540 And the nitrite concentration obtained after conversion. Detailed Implementation

[0051] This invention discloses a method for detecting pentachloronitrobenzene using an enzyme-catalyzed reaction and the Griess reagent method. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the same result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0053] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0054] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0055] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0056] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0057] In one implementation, the enzyme system of the rapid detection method for pentachloronitrobenzene based on enzyme catalysis of the present invention consists of ferrosulfonin protein reductase, ferricredoxin, and nitrobenzene dioxygenase.

[0058] In a specific implementation, each component of the enzyme system is obtained through expression in *E. coli*. In one example, the specific steps include:

[0059] (1) Take competent cells and transform the plasmids of the three enzymes respectively, then plate them and incubate them in a constant temperature incubator overnight;

[0060] (2) Pick colonies from the overnight bacterial plate and transfer them to LB medium. Continue to incubate in a constant temperature shaker (200 rpm, 37°C) until turbidity is reached;

[0061] (3) IPTG was then added and the culture medium was placed in a constant temperature shaker (150 rpm, 24℃) for induction for 14-16 h;

[0062] (4) After induction, centrifuge (5000 rpm, 4℃) for 10 min, discard the supernatant, resuspend the precipitate with buffer and sonicate on ice (280 W, 30 min), then centrifuge again (10000 rpm, 4℃) for 10 min and collect the supernatant to obtain crude enzyme solution.

[0063] (5) The enzyme was purified by using a nickel nucleophilic chromatography column: the crude enzyme solution was loaded onto the membrane and collected by gradient elution with a buffer containing imidazole. Its composition and purity were verified by SDS-PAGE and Native-PAGE.

[0064] In one example, the buffer solution used in steps (4) and (5) above is 50 mM MES with a pH of 7.2.

[0065] In one example, during the induction process in step (3) above, L-cysteine ​​at a final concentration of 0.05 mM and ferrous sulfate at 0.1 mM are added to promote the formation of iron-sulfur clusters.

[0066] In one example, prokaryotic expression was induced by adding IPTG at a final concentration of 0.4 mM, L-cysteine ​​at 0.05 mM, and ferrous sulfate at 0.1 mM, and inducing for 14–16 h in a constant temperature shaker (150 rpm, 24 °C).

[0067] In one example, the molecular weights of the above-mentioned iron sulfoflavin protein reductase (SEQ ID NO: 1), ferroredoxin (SEQ ID NO: 2), and nitrobenzene dioxygenase (α subunit: SEQ ID NO: 3; β subunit: SEQ ID NO: 4) are 35 kDa, 15 kDa, and 225 kDa, respectively.

[0068] In one example, the three enzymes were mixed in a specific ratio, and then NADH with a final concentration of 0.2 mM and an appropriate amount of substrate pentachloronitrobenzene were added. The mixture was reacted in a constant temperature shaker at 35 °C (100 rpm) for 45 min. Then, an appropriate amount of p-aminobenzenesulfonic acid was added and the mixture was reacted for 5 min. Finally, an appropriate amount of naphthylethylenediamine hydrochloride was added and the mixture was reacted for 15 min.

[0069] In one example, the ratio of the above-mentioned ferroflavin protein reductase, ferroredoxin, and nitrobenzene dioxygenase is 1:2:1.

[0070] In one example, the detection limit for the above reaction is 0.0566 mg / kg.

[0071] The amino acid sequences of each component of the enzyme system of the present invention are as follows.

[0072] ① Iron sulfide reductase:

[0073] MELVVEPLNLHLNAETGSTLLDVLRSNEVPISYSCMSGRCGTCRCRVIAGHLRDNGSETGRPQAGKGAYVLACQAVLTEDCTIEIPESDEIVVHPARIVKGTVTAIDEATHDIRRLRIKLAKPLEFSPGQYATVQFTPECVRPYSMAGLPSDAEMEFQIRAVPGGHVSNYVFNELSVGASVRISGPLGTAYLRRTHTGPMLCVGGGTGLAPVLSIVRGALESGMSNPIHLYFGVRSEQDIYDEERLHALAARFPNLKVNVVVATGPAGPGHRSGLVTDLIGRDLPNLAGWRAYLCGAPAMVEALNLLVARLGIVPGHIHADAFYPSGV (SEQ ID NO: 1);

[0074] ② Ferredoxin:

[0075] MSSLSRTVRVRQRDDPQLPHLPHLRLQNMSENWIDAIARDAVPEGDVVGVIVAGKDIAFYEVEGEVFATDNLCTHGAARLSDGFLEGREIECPLHQGRFDVCTGKALCTPLTQDIKTYPVKIENMRVMLKLD (SEQ ID NO:2);

[0076] ③ Nitroarene dioxygenase:

[0077] α subunit:

[0078] MSYQNLVSEAGLTQKLLIHGDKELFQHELKTIFARNWLFLTHDSLIPSPGDYVKAKMGVDEVIVSRQNDGSVRAFLNVCRHRGKTLVHAEAGNAKGFVCGYHGWGYGSNGEL QSVPFEKELYGDAIKKKCLGLKEVPRIESFHGFIYGCFDAEAPPLIDYLGDAAWYLEPTFKYSGGLELVGPPGKVVVKANWKSFAENFVGDGYHVGWTHAAALRAGQSVFSSI AGNAKLPPEGAGLQMTSKYGSGMGVFWGYYSGNFSADMIPDLMAFGAAKQEKLAKEIGDVRARIYRSFLNGTIFPNNSFLTGSAAFRVWNPIDENTTEVWTYAFVEKEMPEDL KRRVADAVQRSIGPAGFWESDDNENMETMSQNGKKYQSSNIDQIASLGFGKDVYGDECYPGVVGKSAIGETSYRGFYRAYQAHISSSNWAEFENASRNWHIEHTKTTDR (SEQ ID NO: 3);

[0079] β subunit:

[0080] MMINTQEDKLVSAHDAEEFHRFFVGHDSDLQQEVTTLLTREAHLLDIQAYKAWLEHFVAPEIKYQVISRELRSTSERRYQLNDAVNLYNENYQQLKVRVEHQMDPQNWANNPKIRFTRFVTNVTAAKDKSAPEILHVRSNLILHRARRENQVDVFYATREDKWKRIEGGGIKLVERFVDYPERIPQTHNLLVFL (SEQ ID NO: 4).

[0081] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0082] The present invention will be further illustrated below with reference to the embodiments.

[0083] Example 1

[0084] Enzyme component acquisition: The preserved bacterial strain was inoculated into 5 mL of LB culture medium containing Kana resistance and activated overnight. After successful activation, the entire strain was inoculated into 500 mL of LB culture medium and cultured in a shaker at 37°C. When the absorbance of E. coli at 600 nm reached approximately 0.8, IPTG (0.4 mM), L-cysteine ​​(0.05 mM), and ferrous sulfate (0.1 mM) were added to a final concentration, and the mixture was induced at 24°C for 16 h.

[0085] After induction, bacterial cells were collected by centrifugation and resuspended in buffer (50 mM MES, pH 6.8). The cells were then sonicated on ice. After sonication, the completely disrupted liquid was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. For purification, the supernatant was transferred to a nickel nucleophilic chromatography column and incubated at 4°C on a shaker for 2–3 h. The nickel column was then washed with buffer (50 mM MES, pH 6.8, 100 mM imidazole), and the eluent was collected. Protein purity was verified by electrophoresis, and the results are shown below. Figure 1 As shown.

[0086] Transformation reaction: The electrophoretically pure enzyme system components were added to a 5 mL tube to bring the final concentrations of ferroflavin protein reductase, feroxin, and nitroaromatic dioxygenase to 30 μM, 60 μM, and 30 μM, respectively. Then, NADH and DMSO were added to dissolve the substrate pentachloronitrobenzene to bring the final concentrations to 0.4 mM and 0.2 mM, respectively. The reaction was then carried out in a shaker (35 ℃, 150 rpm) for 45 min.

[0087] Colorimetric reaction: The conversion reaction of pentachloronitrobenzene produces nitrite ions, therefore the Griess reagent method is used for colorimetric development. Weigh 0.1 g of p-aminobenzenesulfonic acid and dissolve it in 20 mL of pure water, then add approximately 5 mL of hydrochloric acid. Weigh 0.1 g of naphthylethylenediamine hydrochloride and dissolve it in 50 mL of pure water, thus obtaining the two reagents required for the colorimetric reaction. Then, take 1 mL of the above reaction system into a 5 mL tube, add 1 mL of freshly prepared p-aminobenzenesulfonic acid, and react for 5 min. Afterward, add 1 mL of naphthylethylenediamine hydrochloride and react in the dark for 15 min. Observe whether a purple-red compound is formed and measure its ultraviolet absorption intensity at 540 nm. Figure 3 It shows a colorimetric reaction.

[0088] Quantitative analysis: Prepare sodium nitrite solutions of 0.02 mg / L, 0.04 mg / L, 0.08 mg / L, 0.16 mg / L, and 0.2 mg / L, and develop the color using the above-described colorimetric method, then measure A. 540 Plot the standard curve (e.g.) Figure 2 As shown), the A obtained after substituting into the pentachloronitrobenzene conversion reaction was... 540The amount of nitrite can be calculated to quantify the amount of substrate participating in the conversion reaction.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0090] Example 2

[0091] A blank solution identical to the sample matrix was prepared, and 11 consecutive measurements were performed according to the steps described in this invention. The standard deviation (σ) of the UV absorbance values ​​from the 11 measurements was calculated. A series of pentachloronitrobenzene standard solutions with concentrations of 0.2, 0.4, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 mg / kg were prepared, and the measurements were performed using the same method to establish a calibration curve. The curve showed good linearity within the specified concentration range, with the linear equation y = 0.0699x + 0.0517, a correlation coefficient R² = 0.9929, a slope b = 0.0566, and σ = 3.3. Based on the formula LOD = 3.3S / b, the limit of detection (LOD) for pentachloronitrobenzene using this method was calculated to be 0.0566 mg / kg.

[0092] Comparative Example 1

[0093] Same as Example 1, except that L-cysteine ​​and ferrous sulfate are not added during induction.

[0094] The results showed that no purple-red compound was generated after the enzyme component was reacted.

[0095] Comparative Example 2

[0096] Same as Example 1, except that the reaction temperature (20~40℃), reaction time (0~60 min), and buffer pH (6.8~7.4) are different.

[0097] A after reaction under different conditions 540 And the concentration of nitrite obtained after conversion, as follows: Figures 4-6 As shown.

Claims

1. Composition, characterized in that, Comprising: a ferredoxin reductase, a ferredoxin, a nitrophenyl dioxygenase.

2. The composition of claim 1, wherein The amino acid sequence of the ferredoxin reductase is shown as SEQ ID NO: 1, the amino acid sequence of the ferredoxin is shown as SEQ ID NO: 2, the amino acid sequence of the alpha subunit of the nitrophenyl dioxygenase is shown as SEQ ID NO: 3, and the amino acid sequence of the beta subunit of the nitrophenyl dioxygenase is shown as SEQ ID NO:

4.

3. Use of the composition of claim 1 or 2 in detecting pentachloronitrobenzene or in preparing a product for detecting pentachloronitrobenzene.

4. A test reagent, characterized by Comprising the composition of claim 1 or 2.

5. The detection reagent of claim 4, wherein the antibody is a monoclonal antibody. Also comprising Griess reagent.

6. Device, characterized in that Comprising the composition of claim 1 or 2 or the detection reagent of claim 4 or 5.

7. A method for the detection of pentachloronitrobenzene, characterized in that Comprising detection based on any one of the following: i) the composition of claim 1 or 2; ii) the detection reagent of claim 4 or 5; iii) the device of claim 6.

8. The detection method of claim 7, wherein, Comprising: Mixing the sample to be tested with NADH, the composition, mixing with p-aminobenzenesulfonic acid, and then mixing with naphthalene acetic acid dihydrazine hydrochloride to obtain the detection result.

9. The detection method of claim 8, wherein, Comprising: Mixing the sample to be tested with NADH, the composition at 35 ℃ and 100 rpm for 45 min, mixing with p-aminobenzenesulfonic acid for 5 min, and then mixing with naphthalene acetic acid dihydrazine hydrochloride for 15 min to obtain the detection result. The molar ratio of the ferredoxin reductase, the ferredoxin, and the nitrophenyl dioxygenase in the composition is 1:2:

1. The final concentration of the NADH is 0.2 mM.

10. The method of claim 7, wherein the detecting is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. The preparation method of the ferredoxin reductase, the ferredoxin, or the nitrophenyl dioxygenase comprises: S1: culturing an engineering bacterium, wherein the engineering bacterium expresses the ferredoxin reductase, the ferredoxin, and / or the nitrophenyl dioxygenase, to obtain a bacterial liquid; S2: mixing IPTG, L-cysteine, and ferrous sulfate with the bacterial liquid, so that the final concentrations of the IPTG, the L-cysteine, and the ferrous sulfate are 0.4 mM, 0.05 mM, and 0.1 mM respectively, inducing, collecting the supernatant after breaking the bacterial body, obtaining a crude enzyme liquid, purifying, and obtaining the ferredoxin reductase, the ferredoxin, and / or the nitrophenyl dioxygenase.

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