阅读说明：本技术 一种测定植物叶中甲酸脱氢酶活性及其评价的方法 (Method for determining activity of formate dehydrogenase in plant leaves and evaluating formate dehydrogenase in plant leaves ) 是由 苏玉红 何晓红 杨玉霞 李晓娟 于 2020-06-18 设计创作，主要内容包括：本发明公开了一种测定植物叶中甲酸脱氢酶活性及其评价的方法,以高温处理的方式,消除叶提取液中甲酸脱氢酶的活性,根据甲酸的化学性质,采用电位滴定法,通过测定外加甲醛后植物新鲜提取液和失活提取液中甲醛含量的减少值,及外加甲醛前后植物新鲜提取液和失活提取液中酸度变化的差异,进而定量评估植物叶中甲酸脱氢酶的活性大小。实验结果表明,植物质量和提取液比例为1:4时,当甲醛浓度为350 mg·L～(-1)时甲酸脱氢酶活性大小依次为：绿萝>牡丹吊兰>芦荟；当甲醛浓度为500～750 mg·L～(-1)时甲酸脱氢酶活性大小依次为：牡丹吊兰>绿萝,芦荟叶提取液未检测到甲酸脱氢酶活性。(The invention discloses a method for measuring the activity of formate dehydrogenase in plant leaves and evaluating the activity of the formate dehydrogenase in the plant leaves, which eliminates the activity of the formate dehydrogenase in a leaf extracting solution in a high-temperature treatment mode, and quantitatively evaluates the activity of the formate dehydrogenase in the plant leaves by measuring the reduction values of formaldehyde content in a fresh plant extracting solution and an inactivated extracting solution after formaldehyde is added and the difference of acidity change in the fresh plant extracting solution and the inactivated extracting solution before and after the formaldehyde is added by adopting a potentiometric titration method according to the chemical property of formic acid. The experimental result shows that when the ratio of the plant mass to the extraction solution is 1:4, the concentration of the formaldehyde is 350 mg.L ‑1 The activity of the formic acid dehydrogenase is as follows in sequence: scindapsus aureus (Gloiopeltis scindapsus) Roxb>Peony chlorophytum comosum>Aloe vera; when the concentration of formaldehyde is 500-750 mg.L ‑1 The activity of the formic acid dehydrogenase is as follows in sequence: peony chlorophytum comosum>The activity of formate dehydrogenase is not detected in the extract of scindapsus aureus and aloe leaves.)

1. A method for measuring the activity of formate dehydrogenase in plant leaves and evaluating the activity of formate dehydrogenase comprises the following steps: preparing an enzyme inactivation plant extract by a high-temperature treatment mode, and quantitatively evaluating the activity of formate dehydrogenase in plant leaves by measuring the reduction values of the formaldehyde content in the fresh plant leaf extract and the inactivation extract after the addition of formaldehyde and the difference of the acidity changes in the fresh plant leaf extract and the inactivation extract before and after the addition of formaldehyde by adopting a potentiometric titration method according to the chemical properties of formic acid.

2. The method for measuring the activity and evaluation of formate dehydrogenase in plant leaves as claimed in claim 1, wherein the method for measuring the activity and evaluation of formate dehydrogenase in plant leaves is as claimed in claim 3.

3. A method for measuring the activity and evaluation of formate dehydrogenase in plant leaves comprises the following steps:

(1) collecting plant samples in the historic places with fresh air and pollution-free soil in the lawn, garden, wasteland and outside school park, and identifying the plant names by using the flower monarch software;

(2) after the fresh plants in the step (1) are transported back by short distance through ice box preservation, the fresh plants are carefully separated from the soil, quickly and thoroughly washed by deionized water, wiped and accurately weighed;

(3) grinding the plant mass and the volume ratio of the distilled water extractant (g: mL; solid-liquid ratio, g: mL; 1:4, 1:10, 1:20) in the step (2) in a porcelain mortar sufficiently and quickly to extract the effective components in the fresh plant leaves, and transferring the effective components to a precooled 100 mL centrifuge tube;

(4) shaking up the supernatant obtained in the step (3), rapidly centrifuging one part of the supernatant at 0-4 ℃ for later use, placing the other part of the supernatant in a test tube, heating the other part of the supernatant in water at 100 ℃ for 10 min, taking out the supernatant, cooling the supernatant, and centrifuging the supernatant for later use; the result shows that the enzyme in the plant leaf extracting solution is completely inactivated after the plant leaf extracting solution is heated in water at 100 ℃ for 10 min;

(5) sucking 20.00 mL of the fresh or inactivated plant leaf extract centrifuged in step (4) into 4 100 mL centrifuge tubes, and adding 8.00 mL of water and formaldehyde solution (350 mg. L) respectively^-1、500 mg·L^-1、750 mg·mL^-1) Adding magnetons, fully stirring the mixed solution on a magnetic stirrer for 1 min, and recording the pH value of the initial solution; then using the accurately calibrated 0.0250 mol.L^-1The pH value of the mixed solution after complete reaction is tested and recorded by an acidimeter while titration is carried out on the sodium hydroxide solution, and the titration is stopped when the pH value of the mixed solution approaches to about 11; the experiments are all provided with three parallels;

(6) sucking 0.50 mL of the fresh or inactivated plant leaf extract centrifuged in step (4) into 12 cuvettes with plugs, and adding 0 mg. L^-1、350 mg·L^-1、500 mg·L^-1、750 mg·L^-10.20 mL of formaldehyde using solution (three parallel for each concentration), respectively adding 0.50 mL of acetylacetone, fixing the volume of 10.00 mL of distilled water, shaking up, heating in a water bath at 60 ℃ for 15 min, cooling, filtering, diluting, measuring the absorbance value of the filtrate at the wavelength of 414 nm by taking water as a reference, and completing an additional formaldehyde purification experiment;

(7) calculating a fresh extracting solution (m) after the purification reaction is finished according to the standard curve and the variation value of the absorbance in the step (6)₁) And an inactivated extract (m)₂) The concentration reduction value of the medium formaldehyde, namely the reduction amount of the formaldehyde in the theoretical actual extracting solution = the generation amount of the formic acid in the extracting solution; comparing the plant blank experiment results, and calculating the actual value (m) of the content of formic acid in the mixed solution after adding formaldehyde into the fresh extracting solution according to the sodium hydroxide concentration and the volume corresponding to the jump range in the titration curve in the step (5)₃) And the actual value (m) of the formic acid content in the mixed solution after the formaldehyde is added to the inactivated extracting solution₄) The actual value of the formic acid in the plant extract is obtained;

evaluation of Formate dehydrogenase Activity: Δ m₅₌ m_1- m₃Represents the amount of formic acid degradation, Δ m, in the fresh extract due to the action of formate dehydrogenase₆₌ m_2- m₄Representing the difference between the formic acid content of the theoretical inactivated extract and the formic acid content of the determined inactive extract, using the difference between the formic acid content of the fresh extract and the formic acid content of the determined inactive extract₇₌Δm₅-Δm₆So as to measure the activity of formate dehydrogenase in fresh extraction;

the calculation formula of the formic acid content is as follows:

in the formula: v, the numerical value of the volume of the sodium hydroxide standard titration solution consumed by the plant mixed solution, mL; c-concentration of sodium hydroxide, mol. L^-1(ii) a M-molar mass of formic acid, g.mol^-1(M=46.03)

When the ratio of plant mass to extract is 1:4, the concentration of formaldehyde is 350 mg.L^-1The activity of the formic acid dehydrogenase is as follows in sequence: scindapsus aureus (Gloiopeltis scindapsus) Roxb>Peony chlorophytum comosum>Aloe vera; when the concentration of formaldehyde is 500-750 mg.L^-1The activity of the formic acid dehydrogenase is as follows in sequence: peony chlorophytum comosum>The activity of formate dehydrogenase is not detected in the extract of scindapsus aureus and aloe leaves.

4. The method of claim 3, wherein the ratio of plant mass to distilled water extractant volume (solid-liquid ratio, g: mL; 1:4, 1:10, 1:20) is set as the ratio of plant mass to distilled water extractant volume under the protection of low temperature in step (3).

5. The method for inactivating formate dehydrogenase in plant leaves as claimed in claim 3, wherein the heating condition used in said step (4) is 100 ℃ water heating for 10 min.

Technical Field

The invention relates to a method capable of measuring activity of formaldehyde dehydrogenase and evaluation thereof, in particular to a method for measuring activity of formate dehydrogenase in plant leaves by a potentiometric titration method.

Background

Formaldehyde is one of the leading pollutants of indoor air, which seriously harms human health. The common method for removing indoor formaldehyde can be divided into fiber filtration, membrane separation, electrostatic complementation, activated carbon adsorption, ozone sterilization and the like, and some commercialized technologies have the risk problems of complex operation, high cost and possibility of generating secondary pollution due to the characteristics of slow formaldehyde release process, long duration and the like. Compared with other purification technologies, the plant purification technology has the advantages of simple operation, no pollution, low cost and low energy consumption, and can play a role in beautifying and decorating the interior.

The literature research shows that when plants purify formaldehyde, formaldehyde is converted into formic acid under the action of formaldehyde dehydrogenase, and formic acid is converted into CO under the action of formate dehydrogenase₂And H₂O, it is known that formate dehydrogenase plays an important role in the metabolic process of formaldehyde. In the metabolic process of formaldehyde in a plant body, the activity of formate dehydrogenase is enhanced, the reduction of the formate content in the plant body is facilitated, the chemical potential difference of the formaldehyde between air and the plant is increased, and the purification efficiency of the plant on the formaldehyde in the air is further increased. Therefore, the activity of the formate dehydrogenase is one of important indexes for evaluating and predicting the formaldehyde tolerance of plants and screening formaldehyde for efficiently purifying the plants.

Disclosure of Invention

The invention aims to solve the technical problem of providing a simple, rapid, low-cost and high-efficiency method for evaluating the activity of formate dehydrogenase in plant leaves.

The technical scheme adopted by the invention is as follows:

the formate dehydrogenase in plant leaves has poor thermal stability and is inactivated at high temperature. Thus, the activity of formate dehydrogenase in the leaf extract is eliminated by high-temperature treatment to prepare a plant leaf-inactivated extract. And (3) quantitatively evaluating the activity of the formate dehydrogenase in the plant leaves by measuring the difference of acidity changes in the fresh plant leaf extracting solution and the inactivated plant leaf extracting solution before and after formaldehyde addition by adopting a potentiometric titration method.

A method for measuring the activity and evaluation of formate dehydrogenase in plant leaves comprises the following steps:

(1) collecting plant samples in the historic places with fresh air and pollution-free soil in the lawn, garden, wasteland and outside school park, and identifying the plant names by using the flower monarch software;

(2) after the fresh plants in the step (1) are transported back by short distance through ice box preservation, the fresh plants are carefully separated from the soil, quickly and thoroughly washed by deionized water, wiped and accurately weighed;

(3) grinding the plant mass and the volume ratio of the distilled water extractant (g: mL; solid-liquid ratio, g: mL; 1:4, 1:10, 1:20) in the step (2) in a porcelain mortar sufficiently and quickly to extract the effective components in the fresh plant leaves, and transferring the effective components to a precooled 100 mL centrifuge tube;

(4) shaking the supernatant obtained in step (3), centrifuging one part at 0-4 deg.C, heating the other part in 100 deg.C water for 10 min, cooling, and centrifuging. The result shows that the enzyme in the plant leaf extracting solution is completely inactivated after the plant leaf extracting solution is heated in water at 100 ℃ for 10 min;

(5) sucking 20.00 mL of the fresh or inactivated plant leaf extract centrifuged in step (4) into 4 100 mL centrifuge tubes, and adding 8.00 mL of water and formaldehyde solution (350 mg. L) respectively^-1、500 mg·L^-1、750 mg·L^-1) Adding magnetons, fully stirring the mixed solution on a magnetic stirrer for 1 min, and recording the pH value of the initial solution; then using the accurately calibrated 0.0250 mol.L^-1While titrating the sodium hydroxide solution, the pH value of the mixed solution after complete reaction is measured and recorded by an acidimeter, and the titration is stopped when the pH value of the mixed solution is close to about 11. The experiments are all provided with three parallels;

(6) sucking 0.50 mL of the fresh or inactivated plant leaf extract centrifuged in step (4) into 12 cuvettes with plugs, and adding 0 mg. L^-1、350 mg·L^-1、500 mg·L^-1、750 mg·L^-1Using 200 mu L of formaldehyde solution (three are arranged in parallel in each concentration), respectively adding 0.50 mL of acetylacetone, fixing the volume of distilled water to 10.00 mL, shaking up, heating in a water bath at 60 ℃ for 15 min, cooling, filtering, diluting, measuring the absorbance value of the filtrate at the wavelength of 414 nm by taking water as a reference, and finishing an additional formaldehyde purification experiment;

(7) calculating a fresh extracting solution (m) after the purification reaction is finished according to the standard curve and the variation value of the absorbance in the step (6)₁) And an inactivated extract (m)₂) The concentration reduction value of the medium formaldehyde, namely the reduction amount of the formaldehyde in the theoretical actual extracting solution = the generation amount of the formic acid in the extracting solution;

comparing the plant blank experiment results, and calculating the actual value (m) of the content of formic acid in the mixed solution after adding formaldehyde into the fresh extracting solution according to the sodium hydroxide concentration and the volume corresponding to the jump range in the titration curve in the step (5)₃) And the actual value (m) of the formic acid content in the mixed solution after the formaldehyde is added to the inactivated extracting solution₄) The actual value of the formic acid in the plant extract is obtained;

evaluation of Formate dehydrogenase Activity: Δ m₅₌ m_1- m₃Represents the amount of formic acid degradation, Δ m, in the fresh extract due to the action of formate dehydrogenase₆₌ m_2- m₄Representing the difference between the formic acid content of the theoretical inactivated extract and the formic acid content of the determined inactive extract, using the difference between the formic acid content of the fresh extract and the formic acid content of the determined inactive extract₇₌Δm₅-Δm₆So as to measure the activity of formate dehydrogenase in fresh extraction;

the calculation formula of the formic acid content is as follows:

in the formula: v, the numerical value of the volume of the sodium hydroxide standard titration solution consumed by the plant mixed solution, mL; c-concentration of sodium hydroxide, mol. L^-1(ii) a M-molar mass of formic acid, g.mol^-1(M=46.03)

When the ratio of plant mass to extract is 1:4, the concentration of formaldehyde is 350 mg.L^-1The activity of the formic acid dehydrogenase is as follows in sequence: scindapsus aureus (Gloiopeltis scindapsus) Roxb>Peony chlorophytum comosum>Aloe vera; when the concentration of formaldehyde is 500-750 mg.L^-1The activity of the formic acid dehydrogenase is as follows in sequence: peony chlorophytum comosum>The activity of formate dehydrogenase is not detected in the extract of scindapsus aureus and aloe leaves.

Compared with the prior art, the invention has the following advantages:

1. the plant materials used by the method are cheap and easy to obtain, and the method can effectively evaluate the activity of formate dehydrogenase in plant leaves, thereby effectively solving the defects of complicated operation steps, time consumption, high enzyme purification difficulty and the like;

2. the method adopts the acetylacetone color development method to measure the added formaldehyde in the plant leaf extracting solution, thereby effectively solving the defects of complex operation of the technology, expensive required instruments and the like;

3. according to the method, a potentiometric titration method is adopted, and the activity of formate dehydrogenase in plant leaves is quantitatively evaluated by measuring the difference of acidity changes in a fresh plant leaf extracting solution and an inactivated plant leaf extracting solution before and after formaldehyde is added;

4. compared with the traditional enzyme determination method, the method of the invention has the advantages of simplicity, rapidness and high efficiency.

Example 1 evaluation of the Activity of Formaldehyde dehydrogenase in leaves of Paeonia suffruticosa

(1) Preparing to transfer 20.00 mL of fresh or inactivated Paeonia suffruticosa leaf extract into 4 100 mL centrifuge tubes, and respectively adding 8.00 mL of water and formaldehyde solution (350 mg. L)^-1、500 mg·L^-1、750 mg·L^-1) Adding magnetons, stirring the mixture on a magnetic stirrer for 1 min, and recording the pH value of the initial solution. Then using the accurately calibrated 0.0250 mol.L^-1The pH value of the mixed solution after complete reaction is tested and recorded by an acidimeter while titration is carried out on the sodium hydroxide solution, and the titration is stopped when the pH value of the mixed solution approaches to about 11; the experiments are all provided with three parallels;

(2) transferring 0.50 mL of fresh or inactivated Paeonia suffruticosa leaf extract into 12 colorimetric tubes with plugs, and adding 0 mg.L^-1、350 mg·L^-1、500 mg·L^-1、750 mg·L^-10.20 mL of formaldehyde using solution (three parallel for each concentration), respectively adding 0.50 mL of acetylacetone, fixing the volume of 10.00 mL of distilled water, shaking up, heating in a water bath at 60 ℃ for 15 min, cooling, filtering, diluting, measuring the absorbance value of the filtrate at the wavelength of 414 nm by taking water as a reference, and completing an additional formaldehyde purification experiment;

(3) according to the method, the activity of formate dehydrogenase in the peony chlorophytum comosum leaves is quantitatively evaluated by measuring the reduction values of the formaldehyde content in the peony chlorophytum comosum leaf fresh extracting solution and the inactivated extracting solution after formaldehyde is added, and the difference of the acidity change in the peony chlorophytum comosum leaf fresh extracting solution and the acidity change in the peony chlorophytum comosum leaf inactivated extracting solution before and after the formaldehyde is added. The result shows that when the weight of the peony chlorophytum comosum leaves and the ratio of the extraction solution is 1:4, the concentration of the added formaldehyde is 350-750 mg.L^-1The activity of the specific formic acid dehydrogenase is 466-1705 mu g.g^-1FW; when the ratio of the weight of the peony chlorophytum comosum leaves to the extraction solution is 1:10, the concentration of the added formaldehyde is 350-750 mg^-1The activity of the specific formic acid dehydrogenase is 682-231 mu g.g^-1FW; when the weight of the peony chlorophytum comosum leaves and the ratio of the extraction liquid is 1:20, the concentration of the added formaldehyde is 350-750 mg^-1The activity of the specific formic acid dehydrogenase is 255-435 mu g.g^-1FW. When the ratio of the quality of the peony chlorophytum comosum leaves to the extracting solution is 1:10, the activity of formate dehydrogenase in the extracting solution is inversely proportional to the concentration of exogenous formaldehyde.

Example 2 evaluation of Formaldehyde dehydrogenase Activity in scindapsus aureus leaf

Similar to example 1, the difference is that the plants in steps (1) and (2) are changed into the same size and same growth vigor of the scindapsus aureus, and the purification amount of the fresh extract and the purification amount of the inactivated extract of the scindapsus aureus leaves to the added formaldehyde and the change value of the acidity are respectively measured. The result shows that when the mass of the scindapsus aureus leaf and the ratio of the extract is 1:4, the concentration of the added formaldehyde is 350-750 mg.L^-1The activity of the hour formate dehydrogenase is 1248-127 mu g.g^-1FW; when the mass of the scindapsus aureus leaves and the ratio of the extract liquid are 1:10, the concentration of the added formaldehyde is 350-750 mg^-1The activity of the specific formic acid dehydrogenase is 335-0 mu g.g^-1FW; when the mass of the scindapsus aureus leaves and the ratio of the extract liquid are 1:20, the concentration of the added formaldehyde is 350-750 mg^-1The activity of the formate dehydrogenase was 0. mu. g.g^-1FW. When the ratio of the weight of the leaves of scindapsus aureus to the extract was 1:20, no formate dehydrogenase was detected because the formaldehyde-removing effect of the extract was observed as the ratio of the weight of the plants to the extract increased, as shown in example 3, based on the evaluation of the activity of formate dehydrogenase in the leaves of aloe

Similar to example 1, the difference is that the plants in steps (1) and (2) are changed into aloe with the same size and the same growth vigor, and the purification amount of the fresh extract and the purification amount of the inactivated extract of the aloe leaves to the added formaldehyde and the change value of the acidity are respectively measured. The result shows that when the mass of the aloe leaves and the proportion of the extract are 1:4, the concentration of the added formaldehyde is 350-750 mg.L^-1The activity of the specific formic acid dehydrogenase is 58-0 mu g.g^-1FW; when the mass of the scindapsus aureus leaves and the ratio of the extract to the extract are 1:10 and 1:20, the concentration of the added formaldehyde is 350-750 mg^-1Benzoic acidThe dehydrogenase activities were all 0. mu. g.g^-1FW. When the ratio of the mass of the aloe leaves to the extracting solution is 1:10 to 1:20, no formate dehydrogenase is detected, because the effective components for removing formaldehyde in the extracting solution are reduced along with the increase of the ratio of the mass of the plants to the extracting solution, so that the content of the formate dehydrogenase in the plants is reduced, and exogenous formaldehyde is converted into formic acid by other functions.