Fluorescence detection system, detection kit and application thereof, and malic acid detection method

Abstract

The invention relates to the technical field of detection, and discloses a fluorescence detection system, a detection kit and application thereof, and a malic acid detection method. The fluorescence detection system comprises MalR protein and a fluorescence reporter. The invention also discloses a detection kit containing the fluorescence detection system. The invention also discloses application of the fluorescence detection system or the detection kit in malic acid content detection and / or malic acid high-yield strain screening. The invention also discloses a fluorescence detection method which adopts the fluorescence detection system or the detection kit. The method comprises the following steps: mixing MalR protein and a fluorescence reporter, carrying out primary incubation, mixing with a target analyte, carrying out secondary incubation, carrying out fluorescence signal determination, and judging whether malic acid exists in the target analyte or determining the content of malic acid in the target analyte. The fluorescence detection system has the advantages of quick response and simplicity and convenience in operation during fluorescence detection.

Description

Technical Field

[0001] The present invention relates to the technical field of detection, and particularly relates to a fluorescence detection system, a detection kit and its application, and a detection method for malic acid. Background Art

[0002] Malic acid, also known as 2-hydroxybutanedioic acid, has the chemical formula C 4 H 6 O 5 , with a relative molecular mass of 134, and is named because it accounts for 97.2% of the total acid content in apples. There is a chiral carbon atom in the structure of malic acid, so there are three forms: L-malic acid, D-malic acid, and the racemate DL-malic acid which is an equimolar mixture of the two.

[0003] The commonly used detection methods for malic acid mainly include chromatography, enzymatic method and electrochemistry method. High performance liquid chromatography (HPLC) is one of the most commonly used methods for detecting malic acid. This method has high sensitivity and high precision, and is suitable for the detection of complex matrix samples, but there are problems such as long time consumption and many steps. Fluorescence method has the advantages of high sensitivity, good specificity and fast response. The fluorescence method for detecting malic acid has certain application prospects. Therefore, it is feasible and valuable to detect malic acid by fluorescence method. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problem that the existing fluorescence method cannot detect malic acid, and provide a fluorescence detection system, a detection kit and its application, and a detection method for malic acid. The fluorescence detection system has the advantages of fast response and simple operation when performing fluorescence detection.

[0005] To achieve the above purpose, in the first aspect of the present invention, a fluorescence detection system is provided, which includes MalR protein and a fluorescence reporter.

[0006] Preferably, the amino acid sequence of the MalR protein is as shown in SEQ ID NO:2.

[0007] More preferably, the nucleotide sequence of the MalR protein is as shown in SEQ ID NO:3.

[0008] More preferably, the nucleotide sequence of the fluorescence reporter is as shown in SEQ ID NO:1.

[0009] Preferably, the weight ratio of the MalR protein to the fluorescence reporter is 1:0.8 - 1.2.

[0010] Preferably, the MalR protein is obtained by inducing expression by introducing an expression vector into a recombinant strain.

[0011] Further preferably, the expression vector is a pET series plasmid, more preferably pET-24a-MalR.

[0012] Further preferably, the recombinant strain is Escherichia coli, more preferably Escherichia coli BL21(DE3).

[0013] Preferably, the conditions for induced expression include at least: temperature is 20 - 25°C, IPTG concentration is 1.25 - 1.3 mM, and time is 4 - 5 h.

[0014] The second aspect of the present invention provides a detection kit, which contains the fluorescence detection system described in the first aspect above.

[0015] The third aspect of the present invention provides the application of the fluorescence detection system described in the first aspect above or the detection kit described in the second aspect above in the detection of malic acid content and / or the screening of malic acid high-yield strains.

[0016] The fourth aspect of the present invention provides a fluorescence detection method for malic acid, which uses the fluorescence detection system described in the first aspect above or the detection kit described in the second aspect above;

[0017] This method includes the following steps: After mixing the MalR protein and the fluorescence reporter for a primary incubation, then mixing with the target analyte for a secondary incubation and fluorescence signal measurement, to determine whether there is malic acid in the target analyte or to measure the content of malic acid in the target analyte.

[0018] Preferably, the conditions for the primary incubation include at least: avoiding light, temperature is 35 - 37°C, and time is 30 - 35 min;

[0019] The conditions for the secondary incubation include at least: avoiding light, temperature is 35 - 37°C, and time is 40 - 45 min.

[0020] Preferably, the process of measuring the content of malic acid in the target analyte includes: constructing a standard curve of malic acid concentration vs. fluorescence signal, and calculating the content of malic acid in the target analyte based on the fluorescence signal of the target analyte according to the standard curve. Through the above technical solution, the fluorescence detection method provided by the present invention is based on the fact that the MalR protein and the fluorescence reporter can bind together under a low malic acid concentration. As the malic acid concentration increases, the binding activity between the MalR protein and the fluorescence reporter is inhibited. The malic acid content is detected by the change in fluorescence intensity during this process and applied to the detection of malic acid content in the actual fermentation broth. This fluorescence detection system has the advantages of fast response and simple operation during fluorescence detection. Description of the Drawings

[0021] Figure 1It is the structural map of the expression vector plasmid pET-24a-MalR in Preparation Example 1 of the present invention;

[0022] Figure 2 It is the comparison chart of the influence of different induction temperatures on the expression level of MalR protein in Preparation Example 2 of the present invention;

[0023] Figure 3 It is the comparison chart of the influence of different IPTG concentrations on the expression level of MalR protein in Preparation Example 3 of the present invention;

[0024] Figure 4 It is the comparison chart of the influence of different induction times on the expression level of MalR protein in Preparation Example 4 of the present invention;

[0025] Figure 5 It is the comparison chart of fluorescence intensities at different incubation temperatures in Example 2 of the present invention;

[0026] Figure 6 It is the comparison chart of fluorescence intensities at different incubation times in Example 3 of the present invention;

[0027] Figure 7 It is the linear relationship chart between malic acid concentration and fluorescence intensity in Example 1 of the present invention. Detailed implementation manners

[0028] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0029] The first aspect of the present invention provides a fluorescence detection system, which includes MalR protein and a fluorescence reporter. The inventors of the present invention unexpectedly found during the process of fluorescence detection of malic acid content that MalR protein and the fluorescence reporter can bind together when the malic acid concentration is low. As the malic acid concentration increases, the binding activity between MalR protein and the fluorescence reporter is inhibited. The malic acid content is detected by the change in fluorescence intensity during this process and is applied to the detection of malic acid content in actual fermentation broth. This fluorescence detection system has the advantages of fast response and simple operation during fluorescence detection.

[0030] According to the present invention, preferably, the amino acid sequence of the MalR protein is as shown in SEQ ID NO: 2. Further preferably, the nucleotide sequence of the MalR protein is as shown in SEQ ID NO: 3. Further preferably, the nucleotide sequence of the fluorescent reporter is as shown in SEQ ID NO: 1. The inventors found that when the above preferred embodiments are adopted, the response speed and sensitivity of the fluorescence detection system can be further improved, and the accuracy of the detection result can be improved.

[0031] According to the present invention, preferably, the weight ratio of the MalR protein to the fluorescent reporter is 1:0.8 - 1.2, specifically it can be 1:0.8, 1:1, 1:1.2, or any value between the above two ratios. The inventors found that when the above preferred embodiments are adopted, the response speed and sensitivity of the fluorescence detection system can be further improved, and the accuracy of the detection result can be improved.

[0032] The present invention has no strict restrictions on the preparation methods of the MalR protein and the fluorescent reporter. Induced expression, preparation can be carried out by using common methods in the art or entrusted to a professional institution for synthesis. Preferably, the MalR protein is obtained by inducing expression by introducing an expression vector into a recombinant strain. Further preferably, the expression vector is a pET series plasmid, more preferably pET-24a-MalR. Further preferably, the recombinant strain is Escherichia coli, more preferably Escherichia coli BL21(DE3). The inventors found that when the above preferred embodiments are adopted, the yield and quality of the MalR protein can be further improved, thereby improving the response speed and sensitivity of the fluorescence detection system and the accuracy of the detection result.

[0033] According to the present invention, preferably, the conditions for induced expression include at least: the temperature is 20 - 25°C, specifically it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any value between the above two values; the IPTG concentration is 1.25 - 1.3 mM, specifically it can be 1.25 mM, 1.26 mM, 1.27 mM, 1.28 mM, 1.29 mM, 1.3 mM, or any value between the above two values; the time is 4 - 5 h, specifically it can be 4 h, 4.5 h, 5 h, or any value between the above two values. The present invention has no strict restrictions on the process of induced expression, and induced expression can be carried out by using conventional methods in the art. Exemplarily, the following method can be used for the induced expression of the MalR protein:

[0034] (1) Construct recombinant strain R1

[0035] Take 1 tube of E.coli BL21(DE3) competent cells and thaw them on ice. Dissolve the synthesized pET-24a-MalR plasmid powder (GenScript Corporation, Nanjing, China) in sterile water to form a solution of 100 - 105 ng / μL. Take 3 - 4 μL and transfer it into the thawed competent cells (gently flick with fingers), place it on ice for 10 - 15 minutes, then place 2 / 3 - 5 / 6 of it in a water bath at 42 - 43 °C for heat shock for 120 - 125 seconds, immediately take it out of the water bath and put it back on ice for 2 - 5 minutes. Add 800 - 850 μL of LB medium (the components of LB medium are tryptone 10 - 12 g / L, yeast extract 5 - 6 g / L, NaCl 10 - 12 g / L, pH adjusted to 7.0 - 7.2, for solid medium, add 1.5 - 2% (m / v) agar powder, sterilize at 121 - 122 °C for 20 - 25 min; when the temperature drops to about 50 - 55 °C, add kanamycin with a final concentration of 100 - 105 μg / mL). And grow it in a shaking incubator at 37 - 38 °C (200 - 210 rpm) for 35 - 40 minutes. Transfer part or all of it onto an LB agar plate containing 100 - 105 μg / mL kanamycin. Invert the plate and culture it overnight at 37 - 38 °C. Pick monoclonal colonies for colony PCR verification, and preserve the strains with correct colony PCR verification to obtain the recombinant strain R1.

[0036] (2) Induce the expression of MalR protein

[0037] Inoculate the constructed recombinant strain R1 with an inoculum volume of 100 - 105 μL into an LB medium containing 50 - 55 μL of kanamycin solution (concentration 500 - 550 μg / mL), and shake culture at a temperature of 37 - 38 °C and a rotation speed of 220 - 230 rpm for 12 - 13 hours to obtain the seed liquid of recombinant strain R1.

[0038] Inoculate the seed liquid into 100 - 110 mL of LB medium containing 50 - 55 μL of kanamycin solution (concentration 500 - 550 μg / mL) at a ratio of 1 - 1.2:100, and shake culture at a temperature of 37 - 38 °C and a rotation speed of 220 - 230 rpm for 2 - 3 hours until the OD reaches 0.6 - 0.8. Add IPTG (add a final concentration of 0.75 - 2.5 nM) for induction, and continue to culture at a temperature of 20 - 22 °C and a rotation speed of 200 - 210 rpm for 16 - 20 h to obtain the fermentation broth of recombinant strain R1.

[0039] (3) Extraction and purification of MalR protein

[0040] Centrifuge the fermentation broth at 5000 - 6000 rpm for 10 - 12 min to remove the supernatant. Dilute the cell precipitate with PBS and ultrasonically disrupt it at 360 W for 40 - 50 min (on for 5 - 6 s, off for 5 - 6 s, at 35 - 36% power). Then centrifuge at 7500 - 8000 rpm for 30 - 35 min at 4 - 5 °C. Take the supernatant and filter it through a 0.45 μm microporous filter membrane to obtain the crude enzyme solution of MalR protein.

[0041] Pass the crude enzyme solution of MalR protein through a His60 Ni Superflow resin purification column, rinse it with 5 - 6 mL of ultrapure water, then rinse it with 10 - 12 mL of Solution B (50 - 55 mM Tris - HCl buffer, pH 8.0 - 8.2, 25 - 28 mM imidazole, 150 - 155 mM NaCl), and finally elute it with 5 - 6 mL of Solution C (50 - 55 mM Tris - HCl buffer, pH 8.0 - 8.2, 500 - 510 mM imidazole, 150 - 155 mM NaCl). Collect the eluate, and then desalt the eluate using a desalting column GE HiTrap Desalting and elute it with Solution A (50 - 55 mM Tris - HCl buffer, pH 8.0 - 8.2) to obtain the pure enzyme solution of MalR protein.

[0042] The second aspect of the present invention provides a detection kit, which contains the fluorescence detection system described in the first aspect above. Other consumables required for fluorescence detection may also be included in the detection kit. The inventors found that when the detection kit containing the fluorescence detection system of MalR protein and the fluorescence reporter is applied to the detection of malic acid content, it has the advantages of fast response and simple operation, and can be used for the detection of a large number of samples.

[0043] The third aspect of the present invention provides the application of the fluorescence detection system described in the first aspect above or the detection kit described in the second aspect above in the detection of malic acid content and / or the screening of malic acid - high - producing strains. The inventors found that when the fluorescence detection system of MalR protein and the fluorescence reporter is applied to the detection of malic acid content, it has the advantages of fast response and simple operation, and can be used for the primary screening of a large number of samples.

[0044] The fourth aspect of the present invention provides a method for fluorescence detection of malic acid. This method uses the fluorescence detection system described in the first aspect above or the detection kit described in the second aspect above. The method includes the following steps: After mixing MalR protein and a fluorescence reporter for a primary incubation, they are mixed with a target analyte for a secondary incubation and fluorescence signal measurement to determine whether malic acid is present in the target analyte or to measure the content of malic acid in the target analyte. The incubation conditions can be carried out by using the commonly used methods in the art for mixed incubation. Preferably, the conditions for the primary incubation include at least: avoiding light, a temperature of 35 - 37 °C, specifically it can be 35 °C, 36 °C, 37 °C, or any value between the above two values; the time is 30 - 35 min, specifically it can be 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, or any value between the above two values; the conditions for the secondary incubation include at least: avoiding light, a temperature of 35 - 37 °C, specifically it can be 35 °C, 36 °C, 37 °C, or any value between the above two values; the time is 40 - 45 min, specifically it can be 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, or any value between the above two values. The inventors found that when the above preferred implementation mode is adopted, the response speed and sensitivity of the fluorescence detection system can be further improved, and the accuracy of the detection results can be improved.

[0045] According to the present invention, preferably, the detection method further includes: The process of measuring the content of malic acid in the target analyte includes: constructing a standard curve of malic acid concentration vs. fluorescence signal, and calculating the content of malic acid in the target analyte based on the fluorescence signal of the target analyte according to the standard curve. The construction method of the standard curve can be carried out by using the commonly used methods in the art. The standard product can be accurately configured by gradient dilution method, and then fluorescence detection is carried out. Exemplarily, the following method can be used for drawing the standard curve:

[0046] Standard solutions containing malic acid (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) with concentrations of 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, and 12.5 g / L are respectively prepared. The fluorescence intensity of the standard solution is measured using a microplate reader (model Synergy-H1M) at an excitation wavelength of 494 nm and an emission wavelength of 525 nm to draw a standard curve. When a large number of malic acid fermentation broth samples need to be detected, the method of the present invention can be used for rapid detection, which is convenient for optimizing the malic acid fermentation process.

[0047] In the present invention, the target analyte can be any sample that may contain malic acid. For example, the fermentation broth of malic acid-producing bacteria, the reaction solution containing malic acid, and the like. Among them, the fermentation broth containing malic acid can be obtained by fermenting with a strain capable of fermenting to produce malic acid. Exemplarily, the following method can be used to obtain the fermentation broth containing malic acid:

[0048] Inoculate the Aspergillus niger RG0095 (preservation number: CCTCC No. M20221573) on a PDA solid medium and grow at 28 - 30 °C for 5 - 6 days until conidia grow; inoculate the spore suspension into a seed medium (preferably, 1 L of the seed medium contains: 40 - 50 g of glucose, 6 - 8 g of peptone from bacteria, KH 2 PO 4 0.75 - 0.8 g, K 2 HPO 4 0.75 - 0.8 g, MgSO 4 ·7H 2 O 0.1 - 0.2 g, CaCl 2 ·2H 2 O 0.1 - 0.2 g, FeSO 4 ·7H 2 O 5 - 6 mg, NaCl 5 - 6 mg, 1 - 2 mg of anhydrous citric acid, autoclave at 115 - 120 °C for 20 - 30 min;), the final concentration of spores is (2 - 3)×10 6 spores / mL, culture at 28 - 30 °C, 200 - 220 rpm for 20 - 21 h; inoculate the well-grown seed liquid into 50 - 60 mL of a fermentation medium (preferably, 1 L of the fermentation medium contains: 100 - 110 g of glucose, 6 - 8 g of peptone from bacteria, KH 2 PO 4 0.15 - 0.2 g, K 2 HPO 4 0.15 - 0.2 g, MgSO 4 ·7H 2 O 0.1 - 0.2 g, CaCl 2 ·H 2 O 0.1 - 0.2 g, CaCO 3 80 - 85 g, FeSO 4 ·7H 2 O 5 - 6 mg, NaCl 5 - 6 mg, 1 - 2 mg of anhydrous citric acid, autoclave at 115 - 120 °C for 20 - 30 min.), culture at 28 - 30 °C, 200 - 220 rpm for 5 - 6 d;

[0049] Uniformly take 2 - 3 mL of the fermentation broth into a 50 mL EP tube, add an equal volume of 1.5 - 2 M hydrochloric acid solution, and let it stand for 10 - 12 min for sufficient acidification. Uniformly take 2 - 3 mL of the acidified solution into a 2 mL EP tube, centrifuge at 12000 - 13000 rpm for 10 - 12 min, take the supernatant and dilute it 10 - 12 times, and filter it through a 0.22 μm aqueous filter membrane to obtain a fermentation broth containing malic acid diluted 20 - 25 times.

[0050] The present invention will be described in detail below through examples.

[0051] In the following examples, unless otherwise specified, the reagents and raw materials used are all conventional commercially available products.

[0052] Preparation Example 1

[0053] (1) Construct recombinant strain R1

[0054] Take 1 tube of E. coli BL21(DE3) competent cells and thaw them on ice. Dissolve the synthesized pET - 24a - MalR plasmid powder (GenScript Corporation, Nanjing, China) in sterile water to form a solution of 100 ng / μL. Take 3 μL and transfer it into the thawed competent cells (gently flick with fingers), place it on ice for 10 minutes, put 2 / 3 of it in a water bath at 42 °C, perform heat shock for 120 seconds, immediately take it out of the water bath and put it back on ice for 2 minutes. Add 800 μL of LB medium (the components of LB medium are 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, adjust the pH to 7.2, add 1.5% (m / v) agar powder to the solid medium, sterilize at 121 °C for 20 min; wait until the temperature drops to about 55 °C and add kanamycin with a final concentration of 100 μg / mL.). And grow it in a 37 °C shaking incubator (200 rpm) for 40 minutes. Transfer all of it onto an LB agar plate containing 100 μg / mL kanamycin. Invert the plate and culture it overnight at 37 °C. Pick monoclonal colonies for colony PCR verification, and preserve the strains with correct colony PCR verification to obtain recombinant strain R1. The structural map of the expression vector plasmid pET - 24a - MalR is as Figure 1 shown.

[0055] (2) Induce the expression of MalR protein

[0056] Inoculate the constructed recombinant strain R1 with an inoculum size of 100 μL into an LB medium containing 50 μL of kanamycin solution (concentration: 500 μg / mL), and shake - culture it at a temperature of 37 °C and a rotation speed of 220 rpm for 12 hours to obtain a seed solution.

[0057] The seed solution was inoculated into 100 mL of LB medium containing 50 μL of kanamycin solution (concentration: 500 μg / mL) at a ratio of 1:100, and cultured with shaking at 37 °C and 220 rpm for 2 hours until the OD reached 0.6. IPTG (final concentration added: 1 mM) was added for induction, and the culture was continued at 20 °C and 200 rpm for 16 h to obtain the fermentation broth of recombinant strain R1.

[0058] (3) Extraction and purification of MalR protein

[0059] The fermentation broth of recombinant strain R1 was centrifuged at 5000 rpm for 10 min to remove the supernatant. The cell pellet was diluted with PBS and sonicated at 360 W for 40 min (5 s on and 5 s off, 35% power). Then, it was centrifuged at 7500 rpm for 30 min at 4 °C, and the supernatant was filtered through a 0.45 μm microporous membrane to obtain the crude enzyme solution of MalR protein.

[0060] The crude enzyme solution of MalR protein was passed through a His60 Ni Superflow resin purification column, rinsed with 5 mL of ultrapure water, then rinsed with 10 mL of Solution B (50 mM Tris-HCl buffer, pH 8.0, 25 mM imidazole, 150 mM NaCl), and finally eluted with 5 mL of Solution C (50 mM Tris-HCl buffer, pH 8.0, 500 mM imidazole, 150 mM NaCl). The eluate was collected and then desalted using a desalting column GE HiTrap Desalting, and eluted with Solution A (50 mM Tris-HCl buffer, pH 8.0) to obtain the pure enzyme solution of MalR protein.

[0061] Preparation Example 2

[0062] The pure enzyme solution of MalR protein was prepared according to the method of Preparation Example 1, except that the induction temperature after adding IPTG in step (2) was different. MalR protein expression was induced at induction temperatures of 15 °C, 25 °C, 30 °C, and 35 °C respectively. The expression conditions compared with the MalR protein prepared in Preparation Example 1 are as Figure 2 shown.

[0063] Preparation Example 3

[0064] The pure enzyme solution of MalR protein was prepared according to the method of Preparation Example 1, except that the concentration of IPTG added in step (2) was different. MalR protein expression was induced under the conditions that the final concentration of IPTG added was 0.75 mM, 1 mM, 1.25 mM, 1.5 mM, 1.75 mM, 2 mM, 2.25 mM, and 2.5 mM respectively. The specific expression conditions are as Figure 3 shown.

[0065] Preparation Example 4

[0066] The pure enzyme solution of MalR protein was prepared according to the method of Preparation Example 1, except that the culture time after adding IPTG induction in step (2) was different. The MalR protein was induced to express under the conditions of culture times of 4 h, 8 h, 12 h, and 20 h respectively. The expression situation compared with the MalR protein prepared in Preparation Example 1 is as Figure 4 shown.

[0067] Preparation Example 5

[0068] Preparation of Fermentation Broth Containing Malic Acid

[0069] The Aspergillus niger strain (Aspergillus niger RG0095) (preservation number: CCTCC No. M20221573) was inoculated on a PDA solid medium and grown at 28 °C for 5 days until conidia grew; the spore suspension was inoculated into a seed medium (1 L of seed medium contained: 40 g of glucose, 6 g of peptone from bacteria, KH 2 PO 4 0.75 g, K 2 HPO 4 0.75 g, MgSO 4 ·7H 2 O 0.1 g, CaCl 2 ·2H 2 O 0.1 g, FeSO 4 ·7H 2 O 5 mg, NaCl 5 - 6 mg, 1 mg of anhydrous citric acid, autoclaved at 115 °C for 20 min;), the final concentration of spores was 2×10 6 spores / mL, cultured at 28 °C and 200 rpm for 20 h; the well - cultured seed liquid was inoculated into 50 mL of fermentation medium (1 L of fermentation medium contained: 100 g of glucose, 6 g of peptone from bacteria, KH 2 PO 4 0.15 g, K 2 HPO 4 0.15 g, MgSO 4 ·7H 2 O 0.1 g, CaCl 2 ·H 2 O 0.1 g, CaCO 3 80 g, FeSO 4 ·7H 2 O 5 mg, NaCl 5 mg, 1 mg of anhydrous citric acid, autoclaved at 115 °C for 20 min.), cultured at 28 °C and 200 rpm for 5 d;

[0070] Take 2 mL of the fermentation broth evenly into a 50 mL EP tube, add an equal volume of 2 M hydrochloric acid solution, and let it stand for 10 min for sufficient acidification. Then take 2 mL of the acidified solution evenly into a 2 mL EP tube, centrifuge at 12000 rpm for 10 min, take the supernatant and dilute it 10 times, and filter it through a 0.22 μm aqueous filter membrane to obtain a fermentation broth containing malic acid diluted 20 times.

[0071] Example 1

[0072] (1) Construct a standard curve

[0073] Use a microplate reader (model Synergy-H1M) to measure the solution containing only the fluorescent reporter at an excitation wavelength of 494 nm and an emission wavelength of 525 nm. The concentration of the fluorescent reporter in the solution is 500 nM, and the measured fluorescence intensity is 5828.5 a.u., which proves that the fluorescent reporter itself does not produce a fluorescence response, that is, the blank control.

[0074] Add MalR protein to the 500 nM fluorescent reporter solution. The protein binds to the special sequence (TTAATTTAAACAAAAA) in the probe, resulting in a response and the fluorescence intensity rising to 153187 a.u. Divide the above solution containing the fluorescent reporter and MalR protein into five portions, and then sequentially add malic acid standards with ratios of 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, and 12.5 g / L (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.). Use a microplate reader (model Synergy-H1M) to measure the fluorescence intensity at an excitation wavelength of 494 nm and an emission wavelength of 525 nm. Plot the standard curve with the malic acid concentration (g / L) as the abscissa and the fluorescence intensity FL intensity (a.u.) as the ordinate, and obtain the standard curve equation: y = -13761x + 182387, R 2 = 0.9994;

[0075] The linear relationship diagram between the malic acid concentration and the fluorescence intensity is as Figure 7 shown.

[0076] It shows that when malic acid is added, the DNA binding activity of MalR is inhibited, so the fluorescence intensity gradually decreases with the increase of the malic acid concentration.

[0077] (2) Fluorescence detection

[0078] Use the fermentation broth containing malic acid prepared in Preparation Example 4 (the malic acid concentration is 3.5 g / L after being diluted 20 times by liquid phase detection, and the malic acid concentration before dilution is 70 g / L) as the target analyte;

[0079] Mix the fluorescent reporter with the purified MalR protein after Preparation Example 1 (the concentration was detected to be 1.3 μg / μL by Novoprotein BCA Protein Quantification Kit-Box2) at a weight ratio of 1:1. Incubate at 37 °C for 30 min in the dark, then add the target analyte in the same weight ratio as the MalR protein, mix by shaking, incubate at 37 °C for 45 min in the dark, and measure the fluorescence intensity using a microplate reader at an excitation wavelength of 494 nm and an emission wavelength of 525 nm. Substitute the measured fluorescence intensity value into the standard curve equation, and the calculated concentration of malic acid in the target analyte is 3.52 g / L, and the concentration of malic acid before dilution is 70.4 g / L.

[0080] Example 2

[0081] Perform fluorescence detection according to the method of Example 1. The difference is that the secondary incubation time after adding the target analyte in step (2) is different. Fluorescence detection is carried out at secondary incubation times of 0 min, 15 min, 30 min, 45 min, and 60 min respectively. The corresponding measured concentrations of malic acid are -4.66 g / L, 0.55 g / L, 1.7 g / L, 3.52 g / L, and 4.17 g / L. The test results are compared with those of Example 1 as Figure 6 shown. It can be seen from Figure 6 that when the incubation time is 45 min - 60 min, the measured fluorescence intensity tends to be stable and is closer to the actual concentration of malic acid. The incubation time of 45 min - 60 min can be selected for fluorescence detection.

[0082] Example 3

[0083] Perform fluorescence detection according to the method of Example 1. The difference is that the secondary incubation temperature after adding the target analyte in step (2) is different. Fluorescence detection is carried out at secondary incubation temperatures of 27 °C, 32 °C, 37 °C, 42 °C, and 47 °C respectively. The corresponding measured concentrations of malic acid are 4.86 g / L, 12.43 g / L, 3.52 g / L, 1.64 g / L, and 2.89 g / L. The test results are compared with those of Example 1 as Figure 5 shown. It can be seen from Figure 5 that when the incubation temperature is 37 °C, it is closer to the actual concentration of malic acid. The incubation temperature of 37 °C can be selected for fluorescence detection.

[0084] Intraday precision

[0085] 1. Prepare standard solutions of malic acid with concentrations of 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, and 12.5 g / L as test samples;

[0086] 2. Detect by injecting samples at different time periods on the same day according to the test method of Example 1 for 6 times. The test results are shown in Table 1.

[0087] Table 1

[0088] Concentration Detection 1 Detection 2 Detection 3 Detection 4 Detection 5 Detection 6 Mean SD(%) RSD(%) 2.5 g / L 2.483 2.497 2.512 2.489 2.503 2.491 2.496 0.011 0.44 5.0 g / L 4.978 5.017 5.003 4.992 4.985 5.024 5.000 0.019 0.38 7.5 g / L 7.502 7.489 7.523 7.513 7.497 7.508 7.505 0.012 0.16 10.0 g / L 9.987 10.024 10.015 9.973 10.032 9.998 10.006 0.023 0.23 12.5 g / L 12.473 12.517 12.489 12.502 12.491 12.508 12.496 0.016 0.13

[0089] Note: SD is the standard deviation, and RSD is the relative standard deviation.

[0090] Repeatability

[0091] 1. Prepare five standard solutions of malic acid with a concentration of 5 g / L as test samples, numbered 1, 2, 3, 4, and 5.

[0092] 2. Detect the test samples numbered 1, 2, 3, 4, and 5 by injecting samples according to the test method of Example 1. Each sample is measured three times a day, and the average value is taken. The test results are shown in Table 2.

[0093] Table 2

[0094]

[0095] It can be seen from the test results of the above embodiments that the fluorescence detection method provided by the present invention has a rapid response, simple operation, high accuracy of detection results, and good reproducibility when measuring the concentration of malic acid.

[0096] The fluorescence detection method provided by the present invention is based on the fact that MalR protein and the fluorescence reporter can bind together when the concentration of malic acid is low. As the concentration of malic acid increases, the binding activity of MalR protein and the fluorescence reporter is inhibited. The content of malic acid is detected by the change of fluorescence intensity in this process, and it is applied to the detection of the content of malic acid in the actual fermentation broth. This fluorescence detection system has the advantages of rapid response and simple operation during fluorescence detection, high accuracy of detection results, and good reproducibility.

[0097] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A fluorescence detection system, characterized in that: The fluorescence detection system includes MalR protein and a fluorescence reporter.

2. The fluorescence detection system according to claim 1, characterized in that: The amino acid sequence of the MalR protein is shown in SEQ ID NO: 2; Preferably, the nucleotide sequence of the MalR protein is as shown in SEQ ID NO: 3; Preferably, the nucleotide sequence of the fluorescent reporter is as shown in SEQ ID NO:

1.

3. The fluorescence detection system according to claim 1, characterized in that: The weight ratio of the MalR protein to the fluorescent reporter is 1:0.8-1.

2.

4. The fluorescence detection system according to claim 1, characterized in that: The MalR protein is obtained by introducing an expression vector into a recombinant strain for inducing expression; Preferably, the expression vector is a pET series plasmid, preferably pET-24a-MalR; Preferably, the recombinant strain is Escherichia coli, preferably Escherichia coli BL21 (DE3).

5. The fluorescence detection system according to claim 4, characterized in that: The induction expression conditions at least include: temperature of 20-25° C., IPTG concentration of 1.25-1.3 mM, and time of 4-5 h.

6. A detection kit, characterized in that: The detection kit contains the fluorescence detection system according to any one of claims 1 to 5.

7. Use of the fluorescence detection system according to any one of claims 1 to 5 or the detection kit according to claim 6 in detecting malic acid content and / or screening malic acid high-producing strains.

8. A fluorescence detection method for malic acid, characterized in that: The method adopts the fluorescence detection system described in any one of claims 1 to 5 or the detection kit described in claim 6; The method comprises the following steps: mixing the MalR protein and the fluorescent reporter for a first incubation, and then mixing with the target analyte for a second incubation, and measuring the fluorescent signal to determine whether malic acid exists in the target analyte or to measure the content of malic acid in the target analyte.

9. The detection method according to claim 8, characterized in that: The conditions of the one incubation at least include: avoiding light, the temperature is 35-37°C, and the time is 30-35min; The conditions of the secondary incubation at least include: avoiding light, a temperature of 35-37° C., and a time of 40-45 min.

10. The detection method according to claim 8, characterized in that: The process of determining the content of malic acid in the target analyte includes: constructing a standard curve of malic acid concentration and fluorescence signal, and calculating the content of malic acid in the target analyte according to the fluorescence signal of the target analyte based on the standard curve.

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