A method for measuring laccase activity in farmland soil

A technology for the determination of activity and enzyme activity, which is applied in the field of determination of laccase activity in farmland soil. Accelerate the reaction time node and other issues, to achieve the effect of high measurement accuracy and accuracy, high repeatability, and easy operation

Inactive Publication Date: 2016-02-24
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, laccase activity in farmland ecosystem soil is relatively low, generally accounting for 1 / 3-1 / 5 of forest soil
In the process of measuring farmland soil laccase activity, on the one hand, the substrate (ABTS, etc.) is consumed rapidly, and it is difficult to grasp the time node of the enzymatic reaction; It is difficult to control the stability of the solution, which has a great impact on the accuracy and reproducibility of the laccase activity determination results.

Method used

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  • A method for measuring laccase activity in farmland soil
  • A method for measuring laccase activity in farmland soil
  • A method for measuring laccase activity in farmland soil

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Experimental program
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Embodiment 1

[0047] A method for assaying laccase activity in farmland soil, the steps of which are as follows:

[0048] A. Extraction of soil laccase extract

[0049] (1) Select four soil samples of red soil dry land (numbered as sample 1 and sample 2) and paddy and dry land (numbered as sample 3 and sample 4) to be tested in the subtropical red soil hilly area, and take 3g samples (passed through a 2mm sieve) soil samples respectively. Air-dried soil) into a 10ml sterile centrifuge tube, add 6ml of 50mM phosphate buffer (pH=7.1), vortex and mix well, and each sample is parallelized three times.

[0050] Described 50mM phosphate buffer preparation (pH=7.1) method is as follows:

[0051] 50mM sodium dihydrogen phosphate (NaH 2 PO 4 ·H 2 O) stock solution: weigh 8.907gNaH 2 PO 4 2H 2 O is dissolved in sufficient water to make a final volume of 1 L;

[0052] 50mM disodium hydrogen phosphate (Na 2 HPO 4 ) stock solution: weigh 17.907gNa 2 HPO 4 12H 2 O is dissolved in sufficient ...

Embodiment 2

[0072] Accuracy and repeatability of the determination method of laccase activity in farmland soil:

[0073] Weigh 0.0300 g of pure laccase (≥50 units / mg, sigma, product number L2157-10KU) into 1 ml of 50 mM phosphate buffer (pH=7.1), vortex until fully dissolved. Pipette 150, 75, 38, 19, 10 μl of laccase solution into the soil (sample 2 of Example 1) laccase extract solution used to determine the optimum pH, and measure according to ③~④ in the above step B (1) Laccase activity, the original scanning curve is shown in image 3 . According to Mie equation: V=V max ×[S] / (K m +[S]), where V is the reaction speed (indicated by the difference ΔABS of the absorbance value in a certain period of time), K m The value is called the Michaelis constant, V max is the reaction velocity when the enzyme is saturated by the substrate, and [S] is the substrate concentration (by adding the volume V of the laccase solution 0 to indicate), after conversion, 1 / V=(K m / V max )×(1 / [S])+1 / V ...

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Abstract

The invention discloses a method for determining activity of laccase in a farmland soil. Based on the traditional spectrophotometric method for detecting the activity of laccase, a simple, convenient, stable and excellent-reproducibility method for determining the activity of the laccase in the farmland soil is provided by determining the most suitable pH of the activity of soil laccase, the addition proportion of a buffer solution to a primer to extract liquid of the soil laccase, and the scanning time of an ultraviolet spectrophotometer. The method has wide applicability and is suitable for determination on the activity of the laccase in the farmland soil sample, such as a dry land, a paddy field, a paddy-upland field rotation, a vegetable farm, a garden and the like; the operation is simple, convenient and quick; the technical requirements are relatively lower; the method is suitable for carrying out analysis and determination of mass samples.

Description

technical field [0001] The invention belongs to the field of biochemistry and environment, and relates to a method for measuring laccase activity in soil, in particular to a method for measuring laccase activity in farmland soil. It is suitable for scientific research, teaching, agriculture and environmental departments to study the activity of key enzymes in the transformation of organic matter in farmland soil. Background technique [0002] Laccase is a copper-containing polyphenol oxidase. In terrestrial ecosystems, laccase is mainly derived from Basidiomycetes, Deuteromycetes, Ascomycetes and some bacteria in fungi, and its main producer is Basidiomycetes. white rot fungus. The main components of organic matter in soil are lignin, cellulose, and hemicellulose. The decomposition and transformation of these compounds are of great significance to soil carbon sequestration, emission reduction, nutrient release and other ecological functions. Laccase catalyzes the oxidation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
Inventor 陈香碧冯书珍苏以荣
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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