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In situ determination method of enzymatic activity in soil

A determination method and technology for enzyme activity, applied in the field of in situ zymography, can solve the problems of not being able to intuitively reflect the response of enzyme activity, and not being able to truly reflect soil enzyme activity.

Inactive Publication Date: 2018-05-15
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are all destructive sampling of the soil, which cannot truly reflect the soil enzyme activity. In addition, the above methods cannot directly reflect the response of the enzyme activity in the soil.

Method used

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  • In situ determination method of enzymatic activity in soil
  • In situ determination method of enzymatic activity in soil
  • In situ determination method of enzymatic activity in soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] A method for in situ determination of enzyme activity in soil, comprising the following steps:

[0051] Step 1: planting a plant in an inclined root box until the plant develops a root system, wherein the first side of the root box can be opened, and during the growth of the plant, the first side faces the ground; Geotropism, in the process of plant growth, plant roots continuously extend sideways to the first side. During specific implementation, the inclination angle of the root box is α, and α may be any angle between 30° and 60°.

[0052] Step 2: Prepare an enzyme activity fluorescently labeled substrate solution with a concentration of 10-14mM, soak the microporous filter membrane into the enzyme activity fluorescently labeled substrate solution; select the corresponding substrate according to the type of enzyme to be measured .

[0053] Step 3: Open the first side, clean up impurities on the surface of the plant root system, and cover the surface of the root sys...

Embodiment 2

[0083] The difference between this example and Example 1 is that the measurement object is replaced by β-glucosidase. During specific implementation, the substrate is replaced with the substrate corresponding to β-glucosidase, and the enzyme activity fluorescently labeled substrate is 4-methylumbelliferone-β-D-glucoside, M=338.29g / mol. That is to say, the enzyme activity fluorescent labeling substrate is 4-methylumbelliferone-β-D-glucoside, the 4-methylumbelliferone-β-D-glucoside used in this example The concentration is 12mM, and its preparation method is as follows: 1) take 0.4059g of 4-methylumbelliferone-β-D-glucoside, 2) dilute to 100ml with 10mM MES buffer.

[0084] As an alternative embodiment, in order to measure the activity of different soil enzymes (invertase, phosphatase, protease, β-glucosidase, etc.), the substrate in the experiment is modified to the substrate corresponding to the enzyme to be measured.

[0085] The specifications of the reagents and material...

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Abstract

The invention relates to the technical field of in situ zymogram, in particular to an in situ determination method of enzymatic activity in soil. The determination method comprises the following stepsof planting plants in an inclined root box until root systems of plants grow out; preparing an enzymatic activity fluorescence labeling substrate solution; soaking a microporous filtering film into the enzymatic activity fluorescence labeling substrate solution; covering the microporous filtering film onto the surfaces of the root systems; covering a light shielding film above the microporous filtering film; after the first side surface is buckled, downwards putting the first side surface of the root box into a culture box to be cultured; taking out the microporous filtering film; putting themicroporous filtering film under an ultraviolet lamp tube; taking photos by a digital camera in a position right above the film; calculating the enzymatic activity of the corresponding region of themicroporous filtering film by using picture grayscale analysis software. The determination method has the advantages that the accuracy is high; the space and time resolution ratios are high.

Description

technical field [0001] The invention relates to the technical field of in-situ zymography, in particular to an in-situ determination method for enzyme activity in soil. Background technique [0002] So far, the determination of soil enzyme activity has a history of more than 60 years. The common methods include spectrophotometry, fluorescence method, titration method, radioisotope method, etc. The most commonly used methods are spectrophotometry and fluorescence method. However, these methods are all destructive sampling of the soil, which cannot truly reflect the soil enzyme activity. In addition, the above methods cannot directly reflect the response of the enzyme activity in the soil. Contents of the invention [0003] In view of this, the present invention provides an in situ assay method for enzyme activity in soil, which can realize real-time detection of enzyme activity in soil without damaging the plant root system, rhizosphere and soil environment. [0004] In or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 方临川段成娇
Owner NORTHWEST A & F UNIV
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