CN110699423A - Reagent, kit and method for detecting concentration of oxalic acid - Google Patents

Reagent, kit and method for detecting concentration of oxalic acid Download PDF

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CN110699423A
CN110699423A CN201911008116.9A CN201911008116A CN110699423A CN 110699423 A CN110699423 A CN 110699423A CN 201911008116 A CN201911008116 A CN 201911008116A CN 110699423 A CN110699423 A CN 110699423A
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solution
concentration
oxalic acid
reagent
mother liquor
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杨建立
李鹏飞
范伟
何琦瑜
郑绍建
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Zhejiang University ZJU
Yunnan Agricultural University
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Yunnan Agricultural University
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Abstract

The invention relates to a reagent, a kit and a method for detecting the concentration of oxalic acid, belonging to the field of oxalic acid detection; the invention is based on a four-step coupling reaction, utilizes the capability of oxalyl coenzyme A synthetase (AAE3) to degrade oxalate (oxalate) to generate Adenosine Monophosphate (AMP), and provides substrates for the following three reactions in sequence so as to enable the three reactions to continue to occur. The four-step coupling reaction finally causes the reduced coenzyme I (NADH) in the reaction system to be consumed, NADH has an absorption peak at 340nm, and the change of the absorption value at 340nm can indicate the concentration of the degraded oxalic acid. Meanwhile, the reagent and the kit prepared based on the method have low cost, and the method can be used for measuring large-scale samples.

Description

Reagent, kit and method for detecting concentration of oxalic acid
Technical Field
The invention relates to the field of oxalic acid detection, and particularly relates to a reagent, a kit and a method for detecting oxalic acid concentration.
Background
Oxalic acid is a common anti-nutritional factor in food, and is almost entirely derived from food in animals and humans, particularly through ingestion by fruits, leafy vegetables, grains, and legumes. The oxalate metabolic pathway is present in bacteria, fungi and plants, but is absent in vertebrates including humans. Thus, oxalic acid, once ingested, must be excreted through the kidneys, as the gastrointestinal tract is unable to excrete oxalic acid. When food rich in oxalic acid is consumed in large quantities, primary and secondary oxaluria may be induced, thereby weakening kidney function, disturbing glutamate metabolism and reducing blood coagulation. Thus, excessive oxalate intake can lead to a range of kidney-related disorders. Meanwhile, the existence of oxalic acid can reduce the absorption of calcium ions in the intestinal tract, so that calcium oxalate precipitates are formed, and finally kidney stones are caused; furthermore, excessive amounts of oxalic acid ingested can form insoluble salts with iron, magnesium, and many trace elements, thereby impairing the utilization of these elements by the human body.
At present, the detection means of oxalic acid comprises high performance liquid chromatography, ion chromatography and the like, but instruments needed in the detection process are expensive and long in time, and meanwhile, the defects of interfering ions, low sensitivity and the like exist.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a reagent, a kit and a method for rapidly detecting the concentration of oxalic acid, which have the advantages of low cost and high sensitivity, have wide application range, and can be used for measuring the content of oxalic acid in body fluids and leaching liquor of various water bodies, animals, plants and microorganisms.
In order to achieve the purpose, the technical scheme of the invention is as follows: reagent for detecting concentration of oxalic acidThe method is characterized by comprising the following steps: 100mM Tris-HCl buffer solution (pH8.0), 1M CoA solution, 5M ATP solution, 1M MgCl2Solution, 400mM NADH solution, 100mM PEP solution, 2KU myokinase, 50mg PEPK, 25KU LDH, 0.5mg/mLAAE 3.
Further, the MgCl2The solution is a cofactor for the enzymatic reaction.
Further, the Tris-HCI buffer solution is used for maintaining the pH value of the reaction system to be stable.
Further, the kit for rapidly detecting the concentration of oxalic acid comprises a plurality of reagent bottles, wherein each reagent bottle is internally provided with a reagent, and the reagents comprise a Tris-HCI buffer solution with the concentration of 100mM and the pH value of 8.0, a 1M CoA solution mother solution, a 5M ATP solution mother solution and a 1M MgCl solution2Solution mother liquor, 400mM NADH solution mother liquor, 100mM PEP solution mother liquor, and 2KU myokinase, 50mg PEPK, 25KU LDH and 0.5mg/mL AAE 3;
at least one bottle of reagent bottle comprises Tris-HCI buffer solution with the concentration of 100mM and pH8.0; at least one bottle of reagent bottle comprises CoA solution mother liquor with the concentration of 1M; at least one bottle of reagent bottle comprises ATP solution mother liquor with the concentration of 5M; at least one bottle of reagent bottle comprises MgCl with the concentration of 1M2Solution mother liquor; at least one bottle of reagent bottle comprises NADH solution mother liquor with the concentration of 400mM, and at least one bottle of reagent bottle comprises PEP solution mother liquor with the concentration of 100 mM; at least one vial of reagent comprises myokinase at a concentration of 2 KU; at least one vial of reagent comprises 50mg of PEPK; at least one reagent bottle comprising LDH at a concentration of 25 KU; at least one bottle of reagent bottle includes a concentration of 0.5mg/mL AAE 3.
Further, a method for detecting the concentration of oxalic acid comprises the following steps:
(1) taking a row of enzyme standard tubes, numbering the enzyme standard tubes, and respectively and sequentially adding a series of oxalic acid solutions with volume gradients;
(2) adding 955 mu of LTris-HCl buffer solution and 10 mu of LMgCl into a centrifuge tube in sequence2The solution mother liquor, 0.5. mu.L of CoA solution mother liquor, 5. mu.L of ATP solution mother liquor, 10. mu.L of NADH solution mother liquor, 10. mu.L of PEP solution mother liquor were gently blown with a 1-ml pipette tipMixing uniformly; respectively adding 10U of myokinase, 10U of PEPK, 10U of LDH and 1 mu of LAAE3 into the uniformly mixed reaction system, and lightly blowing and uniformly mixing the mixture by using a 1ml gun head;
(3) respectively and sequentially adding the uniformly mixed reaction systems in the step (2) into the enzyme-labeled tubes numbered in the step (1), and lightly shaking and mixing to make the mixed volume be 100 mu L, thus obtaining a series of oxalic acid standard solutions with concentration gradient;
(4) numbering three enzyme-labeled tubes, respectively adding substrates to be detected with different volumes, supplementing the mixture to 100 mu L by using the uniformly mixed reaction system in the step (2), and slightly shaking;
(5) measuring the light absorption value of each enzyme label tube at 340nm in the step (4) by using an enzyme label instrument, and simultaneously reading 4 data of each enzyme label tube for 3 times continuously; after reacting for 10min, measuring the light absorption value at 340nm in the same way again;
(6) and (3) drawing a standard curve by taking the concentration of the oxalic acid standard solution as an abscissa and the difference of absorbance before and after NADH reaction as an ordinate, fitting a linear equation, and bringing the difference of absorbance before and after the sample solution to be measured into the linear equation to calculate the concentration of oxalic acid in the sample solution to be measured.
Further, if the absorbance of the sample solution to be measured in step (6) is out of the range of the standard curve, the sample needs to be further diluted or concentrated.
Further, it is characterized in that: the oxalyl-coa synthetase AAE3 is an enzyme that has been subjected to induction purification.
Further, the oxalyl-CoA synthetase AAE3 induced purification steps are as follows:
(1) introducing the gene coding oxalyl-coenzyme A synthetase in tomato into escherichia coli through plasmids, and inducing by using galactosyl-anhydrase IPTG; (2) then nickel column purification is used for inducing protein, so that oxalyl coenzyme A synthetase is enriched and purified;
furthermore, color developers 1-methoxy-5-methyl-phenazine methyl sulfate and tetrazole nitro blue are added into the reaction system. When oxalic acid is absent, the color is purple, and when oxalic acid is present, the purple becomes lighter due to the reaction.
Further, the detection limit of the oxalic acid concentration is 1 mu mol/L.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting the concentration of the oxalic acid is short in time consumption, the detection can be completed within 10min, the requirement on the sample amount is low, and the sample amount is less than 10 mu L when the sample is detected; meanwhile, the sensitivity is high, the linear range is wide, the kit is suitable for 1-100 mu M, the detection limit of oxalic acid is 1 mu M, and is far lower than 20 mu M of a commercially available sigma kit; meanwhile, an enzyme AAE3 with higher affinity to oxalic acid is found, and the detection limit of oxalic acid in a sample can be obviously improved by using the enzyme; secondly, the AAE3 is obtained by adopting a prokaryotic expression and 6 × His affinity purification method, so that the purity of the enzyme is improved, and the obtaining cost is greatly reduced;
the color-developing agents 1-methoxy-5-methyl-phenazine methyl sulfate and tetrazole nitro blue are added in the detection process, so that the method can be used for qualitative detection, can be identified by naked eyes, and is quick and convenient.
The cost of the kit prepared by the reagent is low, the cost of a single reaction is only 1.46 yuan, and the cost is only 1/93 of a commercially available sigma kit and is far lower than that of the commercially available sigma kit, and the kit can be used for measuring large-scale samples.
Drawings
FIG. 1 is a graph of the standard curve of the present invention;
FIG. 2 is a map of the plasmid pET28a by two-enzyme cleavage;
FIG. 3 shows PCR positive identification of transformed E.coli;
FIG. 4 is a prokaryotic inducible expression diagram of SlAAE 3;
FIG. 5 SlAAE3 His affinity purification conditions diagram;
FIG. 6 shows the effect of concentration of SlAAE3 protein;
FIG. 7 SlAAE3 substrate specificity chromogenic map.
Detailed Description
The technical solution of the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
A reagent for detecting the concentration of oxalic acid, comprising: Tris-HCl buffer solution of 100mM pH8.0CoA solution at a concentration of 1M, ATP solution at a concentration of 5M, and MgCl at a concentration of 1M2Solution, 400mM NADH solution, 100mM PEP solution, 2KU myokinase, 50mg PEPK, 25KU LDH, 0.5mg/mL AAE 3.
Example 2
A kit for rapidly detecting the concentration of oxalic acid, the kit comprising a plurality of reagent bottles, each reagent bottle containing a reagent, the reagent comprising: 100mM Tris-HCl buffer solution (pH8.0), 1M CoA solution, 5M ATP solution, 1M MgCl2Solution, 400mM NADH solution, 100mM PEP solution, 2KU myokinase, 50mg PEPK, 25KU LDH, 0.5mg/mL AAE 3.
Example 3
A method for detecting the concentration of oxalic acid is based on four-step coupling reaction:
Figure BDA0002243365830000062
Figure BDA0002243365830000063
Figure BDA0002243365830000064
the method comprises the following steps:
(1) preparing 1mM oxalic acid solution by using Tris-HCl buffer solution in the kit, and weighing 90.04mg oxalic acid by using an electronic balance to dissolve in 1L Tris-HCl solution to obtain 1mM (1mmol/L) oxalic acid solution; 90.04mg of oxalic acid can also be weighed and dissolved in 1mL of Tris-HCl solution to obtain 1M (1mol/L) of oxalic acid solution (which can be stored at room temperature), and diluted according to the amount of the oxalic acid solution required by the experiment, for example, 1mL of oxalic acid solution is required, and 1 microliter of 1M of oxalic acid solution is added into 999 microliter of Tris-HCl solution to obtain 1mL of oxalic acid solution with the concentration of 1 mM.
Then taking a row of enzyme standard tubes and numbering 1-7, and respectively and sequentially adding 0, 0.5, 1, 2, 5, 8 and 10 mu L of oxalic acid solution;
(2) a2 mL centrifuge tube was sequentially charged with 955. mu.L Tris-HCl solution and 10. mu.L MgCl2Mother liquor, 0.5 mu L of CoA mother liquor, 5 mu L of LATP mother liquor, 10 mu L of NADH mother liquor and 10 mu L of PEP mother liquor are lightly blown and beaten by a 1ml gun head and evenly mixed; respectively adding 10U of myokinase, PEPK, LDH (the adding volume is calculated according to the actual enzyme activity in the kit) and 1 muL of AAE3 (the final concentration after adding glycerol) into the uniformly mixed reaction system, and slightly blowing and beating the uniformly mixed reaction system by using a 1mL gun head;
(3) sequentially adding 100, 99.5, 99, 98, 95, 92 and 90 mu L of the uniformly mixed reaction system in the step (2) into the enzyme labeling tubes numbered 1 to 7 in the step (1), and lightly shaking to obtain standard solutions with oxalic acid concentrations of 0, 5, 10, 20, 50, 80 and 100 mu M;
(4) numbering another 3 enzyme-labeled tubes, respectively adding 1, 5 and 10 mu L of the substrate to be detected, adding the substrate to 100 mu L by using the uniformly mixed reaction system in the step (2), and slightly shaking;
(5) measuring the light absorption value of each enzyme label tube at 340nm in the step (4) by using an enzyme label instrument, and simultaneously reading 4 data of each enzyme label tube for 3 times continuously; after reacting for 10min, measuring the light absorption value at 340nm in the same way again;
(6) the concentration of oxalic acid standard solution is used as abscissa, the difference of absorbance before and after NADH reaction is used as ordinate to draw a standard curve, a linear equation is fitted, as shown in figure 1, the measurement has a linear relation in the concentration range of 1-100 μ M, R20.998, a good linear relationship; and (4) bringing the difference of the absorbances of the solution of the sample to be detected into a linear equation to calculate the concentration of the oxalic acid in the solution of the sample to be detected. The light absorption value of oxalic acid in the sample solution to be detected is the best value in the middle of the standard curve, if the light absorption value exceeds the upper limit of the range of the standard curve, the sample needs to be further diluted, and if the light absorption value exceeds the lower limit of the range of the standard curve, the sample needs to be further concentrated.
The oxalyl coenzyme A synthetase AAE3 is an enzyme subjected to induced purification, and the steps of induced purification are as follows:
(1) introducing the gene coding oxalyl-CoA synthetase in tomato into escherichia coli through plasmids, and inducing by using galactosyl-anhydrase IPTG, specifically comprising the following steps:
extracting total tomato RNA, carrying out reverse transcription to obtain tomato cDNA, designing a primer according to CE Design V1, and amplifying the full length of a SlAAE3(Solyc03g025720.2.1) gene; carrying out double digestion on pET28a (+) plasmid by using BamH I and Sal I, connecting with SlAAE3, transferring into DH5 alpha strain, picking positive clone and sequencing, wherein the result is shown in a figure 2 and a figure 3;
selecting the strain with the brightest band in the figure 3 for carrying out the subsequent protein induction experiment, transferring the plasmid with the correct sequencing result into a BL21 strain, inoculating the strain into an LB liquid culture medium containing 100 mu g/mL kanamycin, shaking at 37 ℃ at 220r/min until the strain is obviously turbid, sucking 200 microliters of bacterial liquid, inoculating the strain into 20mL of LB liquid culture medium containing 100 mu g/mL kanamycin, and shaking at 37 ℃ at 220r/min until the OD600 is 0.4-0.8; dividing the bacterial liquid into 2 parts, each part is 10mL, adding IPTG into one part to enable the final concentration to be 0.5mM, and shaking at 16 ℃ for 110r/min or 200r/min for 8-10 h; centrifugally collecting thalli, resuspending the thalli by PBS, centrifuging again after ultrasonic crushing, and separating precipitates and supernatant; adding 5 Xloading buffer in proportion, carrying out polyacrylamide gel electrophoresis at 95 ℃ for 10min, and dyeing with Coomassie brilliant blue, wherein the result is shown in the comparison of bands of a control group CK and an IPTG group in a figure 4, and the result shows that after the IPTG is added, the target protein is successfully induced and expressed, and both the sediment and the supernatant contain the target protein;
(2) then nickel column purification is used for inducing protein, so that oxalyl coenzyme A synthetase is enriched and purified;
sealing the bottom of the column, adding 2ml of Ni NTA beads into the column, naturally standing until the beads are separated from the protective solution, and opening the lower end seal of the column to enable the protective solution to naturally flow out; adding 10ml of lysine buffer to soak and wash the beads, and dividing the mixture into two times, wherein each time is 5 ml; adding the strain cracking supernatant into beads, and collecting effluent liquid; the column was rinsed with 20ml of wash buffer containing 20mM imidazole and the effluent collected; the column was rinsed with 20ml of wash buffer containing 50mM imidazole and the effluent collected; the column was rinsed with 20ml of wash buffer containing 100mM imidazole and the effluent collected; the column was rinsed with 800. mu.L of an elution buffer, repeated 4 times, and the effluent collected and labeled E1-4, respectively. Taking a proper amount of the collected effluent, adding a corresponding proportion of 5 × loading buffer, carrying out polyacrylamide gel electrophoresis at 95 ℃ for 10min, and dyeing with Coomassie brilliant blue, wherein the result is shown in FIG. 5, and the result shows that the content of the target protein in the effluent is very small, which indicates that the target protein is successfully adsorbed by NTA beads; the band of the target protein in the lane 20wash is very weak, which indicates that 20mM of imidazole cannot elute the target protein; when the concentration of imidazole is more than 50mM, the target protein is successfully eluted; then combining the effluent containing the target protein together, and concentrating by using a 50000Da molecular sieve, wherein the concentration of the concentrated protein is increased as shown in figure 6, and then adding glycerol with the same volume into the purified AAE3 enzyme solution for mixing, wherein the glycerol has the function of protecting the activity of the AAE3 enzyme, so that the protected protein is not easy to degrade and denature;
furthermore, color developers 1-methoxy-5-methyl-phenazine methyl sulfate and tetrazole nitro blue are added into the reaction system. In the absence of oxalic acid, the color was purple, and if the reaction occurred, the color became lighter, as shown in FIG. 7; based on this, can be used as the basis of qualitative analysis.
Comparative example 1 Sigma oxalic acid detection kit
The Sigma oxalate kit is numbered MAK315, the storage conditions are-20 ℃, and the kit contains 100 microliters of Reagent A (numbered MAK307A), 18 milliliters of Reagent B (numbered MAK307B), 1 milliliter of oxalate standard (the concentration is 500 micromole per liter), 120 microliters of HRP enzyme (numbered MAK307D) and 120 microliters of oxalate oxidase (numbered MAK 307E). The specific operation steps are as follows:
1. 10 microliter samples were taken and added to 3 independent centrifuge tubes, with the 3 spaces being blank, sample and internal standard, respectively.
2. 10 microliters of ultrapure water was added to the blank and the sample, respectively, and 10 microliters of oxalic acid standard was added to the internal standard.
3. Denaturation (for urine samples; non-urine samples directly into step 4). Dissolve 5. mu.l of reagent A in 20ml of water. Aspirate 30 microliters of diluted reagent a, add to the above 3 sample tubes, tap the tube walls and mix well, and stand at room temperature for 2 minutes.
4. Preparing a working solution: for the blank: 155 microliters of reagent B and 1 microliter of HRP enzyme were mixed well. For the samples and internal standards: take 155. mu.l of reagent B, 1. mu.l of oxalate oxidase and 1. mu.l of HRP enzyme.
Note: the working solution can be stored for 2 hours, and is recommended to be prepared for use.
5. Add 150 μ l of blank solution to the blank tube, add 150 μ l of working solution to the sample tube and the internal standard tube, respectively, and mix well.
6. After standing at room temperature for 10 minutes, the absorbance was read at 595 nm.
And (4) calculating a result:
the concentration of oxalic acid in the sample was calculated as follows:
note: ODSAMPLE，ODSTANDARD，ODBLANKRefer to the absorbance at 595nm for the sample, internal standard and blank, respectively. 500 is the concentration of oxalic acid in the internal standard and n is the dilution factor of the sample. If the measured concentration of the oxalic acid in the sample is more than 1000 micromole/liter, the sample needs to be further diluted, the measurement is repeated, and finally the real concentration of the oxalic acid in the sample is obtained by multiplying the dilution times. Compared with a sigma product, the product has the advantages that:
1. the oxalyl coenzyme A synthetase SlAAE3 in the tomato adopted by the product has stronger affinity to oxalic acid and lower detection limit, which can reach 1 mu mol/L and is far lower than the detection limit of 20 mu mol/L of sigma company;
the sigma product is sold at 5451.28 RMB, the maximum 120 reactions can be theoretically carried out, and 40 samples can be measured according to the measuring scheme provided in the kit, namely the cost of a single sample is about 136 yuan. The cost of the kit can be divided into two parts: the statistics of the table 1 show the approximate cost of the construction of the prokaryotic expression strain, and the cost of the link can be used for a long time of 86.69 yuan; table 2 shows the statistics of the cost of AAE3 protein induction and reaction system construction, because the AAE3 protein induced by a single time is probably enough for 1000 reactions, the total cost of 1000 reactions is firstly counted, and then the cost of a single time is calculated to be about 1371.22 yuan. Combining the total cost of the two parts, the cost of a single reaction of the kit is about 1.46 yuan.
Table 1: cost statistics of target gene acquisition and expression bacterium construction links
Figure BDA0002243365830000111
Table 2: cost statistics of AAE3 protein induction and reaction system
Figure BDA0002243365830000121
And 3, the experimental scheme provided by the sigma product adopts an internal standard method for quantification, and is more suitable for small-scale sample determination. The standard curve adopted by the product is quantitative, and the method is more suitable for measuring large-scale samples.
The above description is only for the preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any changes and substitutions without any inventive work on the basis of the present invention should be covered by the protection scope of the present invention, and therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims (10)

1. A reagent for detecting the concentration of oxalic acid, comprising: 100mM Tris-HCl buffer solution (pH8.0), 1M CoA solution, 5M ATP solution, 1M MgCl2Solution, 400mM NADH solution, 100mM PEP solution, 2KU myokinase, 50mg PEPK, 25KU LDH, 0.5mg/mL AAE 3.
2. The reagent according to claim 1, characterized in that: said MgCl2The solution is a cofactor for the enzymatic reaction.
3. The reagent according to claim 1, characterized in that: the Tris-HCI buffer solution is used for maintaining the pH value of the reaction system to be stable.
4. A kit for detecting the concentration of oxalic acid, prepared based on the reagent of claim 1, characterized in that: the kit comprises a plurality of reagent bottles, wherein each reagent bottle is internally provided with a reagent, and the reagent comprises a Tris-HCI buffer solution with the concentration of 100mM and the pH value of 8.0, a CoA solution mother solution with the concentration of 1M, an ATP solution mother solution with the concentration of 5M and MgCl with the concentration of 1M2Solution mother liquor, 400mM NADH solution mother liquor, 100mM PEP solution mother liquor, and 2KU myokinase, 50mg PEPK, 25KU LDH and 0.5mg/mL AAE 3;
at least one bottle of reagent bottle comprises Tris-HCI buffer solution with the concentration of 100mM and pH8.0; at least one bottle of reagent bottle comprises CoA solution mother liquor with the concentration of 1M; at least one bottle of reagent bottle comprises ATP solution mother liquor with the concentration of 5M; at least one bottle of reagent bottle comprises MgCl with the concentration of 1M2Solution mother liquor; at least one bottle of reagent bottle comprises NADH solution mother liquor with the concentration of 400mM, and at least one bottle of reagent bottle comprises PEP solution mother liquor with the concentration of 100 mM; at least one vial of reagent comprises myokinase at a concentration of 2 KU; at least one vial of reagent comprises 50mg of PEPK; at least one reagent bottle comprising LDH at a concentration of 25 KU; at least one bottle of reagent bottle includes 0.5mg/mL AAE3 concentration.
5. A method for detecting the concentration of oxalic acid, comprising the steps of:
(1) taking a row of enzyme standard tubes, numbering the enzyme standard tubes, and respectively and sequentially adding a series of oxalic acid solutions with volume gradients;
(2) adding 955 mu of LTris-HCl buffer solution and 10 mu of LMgCl into a centrifuge tube in sequence2The solution mother liquor, 0.5 mu L of CoA solution mother liquor, 5 mu L of ATP solution mother liquor, 10 mu L of NADH solution mother liquor and 10 mu L of PEP solution mother liquor are lightly blown and uniformly mixed by a 1mL gun head; respectively adding 10U of myokinase, 10U of PEPK, 10U of LDH and 1 mu L of AAE3 into the uniformly mixed reaction system, and lightly blowing and uniformly mixing the mixture by using a 1mL gun head;
(3) respectively and sequentially adding the uniformly mixed reaction systems in the step (2) into the enzyme-labeled tubes numbered in the step (1), and lightly shaking and mixing to make the mixed volume be 100 mu L, thus obtaining a series of oxalic acid standard solutions with concentration gradient;
(4) numbering three enzyme-labeled tubes, respectively adding substrates to be detected with different volumes, supplementing the mixture to 100 mu L by using the uniformly mixed reaction system in the step (2), and slightly shaking;
(5) measuring the light absorption value of each enzyme label tube at 340nm in the step (4) by using an enzyme label instrument, and simultaneously reading 4 data of each enzyme label tube for 3 times continuously; after reacting for 10min, measuring the light absorption value at 340nm in the same way again;
(6) and (3) drawing a standard curve by taking the concentration of the oxalic acid standard solution as an abscissa and the difference of absorbance before and after NADH reaction as an ordinate, fitting a linear equation, and bringing the difference of absorbance before and after the sample solution to be measured into the linear equation to calculate the concentration of oxalic acid in the sample solution to be measured.
6. An oxalic acid concentration detection method according to claim 5, characterized in that: and (4) if the light absorption value of the sample solution to be detected in the step (6) exceeds the range of the standard curve, further diluting or concentrating the sample.
7. An oxalic acid concentration detection method according to claim 5, characterized in that: the oxalyl-coa synthetase AAE3 is an enzyme that has been subjected to induction purification.
8. An oxalic acid concentration detection method according to claim 7, characterized in that: the oxalyl-CoA synthetase AAE3 induction purification steps are as follows:
(1) introducing the gene coding oxalyl-coenzyme A synthetase in tomato into escherichia coli through plasmids, and inducing by using galactosyl-anhydrase IPTG;
(2) then nickel column is used for purifying the induced protein, so that the oxalyl coenzyme A synthetase is enriched and purified.
9. The method according to any one of claims 5-8, wherein: adding color-developing agents 1-methoxy-5-methyl-phenazine methyl sulfate and tetrazole nitro blue into a reaction system; in the absence of oxalic acid, the color is purple, with oxalic acid, the reaction takes place, and the purple color becomes lighter.
10. The method of claim 9, wherein: the detection limit of the oxalic acid concentration is 1 mu mol/L.
CN201911008116.9A 2019-10-22 2019-10-22 Reagent, kit and method for detecting concentration of oxalic acid Pending CN110699423A (en)

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Application publication date: 20200117