Method for extracting soil mircoorganism carbon-sequestrated enzyme

A soil microbiology and soil technology, which is applied in the determination/inspection of microbes, biochemical equipment and methods, lyase, etc., can solve the problems of lack of systematic research and uninvolved issues, and achieve sensitivity improvement, pollution reduction, precision and The effect of high accuracy

Inactive Publication Date: 2012-07-04
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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Problems solved by technology

However, the current research has not involved the extraction and activity determination of RubisCO in environmental samples, especially the relationship between the activity of RubisCO 1,5-bisphosphate carboxylase / oxygenase (RubisCO) and the carbon fixation ability of environmental microorganisms is lacking. systematic research

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  • Method for extracting soil mircoorganism carbon-sequestrated enzyme
  • Method for extracting soil mircoorganism carbon-sequestrated enzyme

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Embodiment 1

[0048] Soil microbial carbon fixation key enzyme 1, the extraction and activity assay method of 5-ribulose diphosphate carboxylase / oxygenase (RubisCO) enzyme, its steps are as follows:

[0049] Weigh 5 grams of sample soil in a 50 ml sterile centrifuge tube, add 25 ml of pre-cooled soil various ions, inorganic matter, and organic extracellular free protein remover (TENP buffer), vortex mix, and then Centrifuge at 10,000×g for 10 minutes at room temperature to collect the precipitate. Repeat this step twice; then add 25 ml of pre-cooled phosphate buffer solution (0.01 mol / L, pH 7.4), vortex and mix well, centrifuge at room temperature at 10,000×g for 10 minutes, collect the precipitate, and repeat this step twice; Finally, 25 ml of sterile ultra-clean water was added, vortexed, centrifuged at 10,000×g for 10 minutes at room temperature, and the precipitate was collected. The precipitate is the extracted crude protein precipitate. The precipitate was freeze-dried and stored at...

Embodiment 2

[0074] use 14 C-CO 2 Continuous labeling technology to explore the effects of light and shading on the activity of the key enzyme 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) in soil microbial carbon fixation and its relationship with the amount of carbon fixation:

[0075] Four typical upland and paddy soils in the subtropical region were selected respectively, and the dry soil and paddy soil were treated with bare soil and shading, and each treatment was repeated 4 times. Apply carbon isotopes ( 14 C) Continuous tracer technology, through an indoor airtight simulation system, to explore the activity of the key enzyme 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) in soil microbial carbon fixation and its relationship with carbon fixation ( 14 C-SOC) relationship. Sampling after marking culture 80d, adopt method of the present invention to measure soil microbial carbon fixation key enzyme 1, 5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) ac...

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Abstract

The invention discloses a method for extracting a soil mircoorganism carbon-sequestrated enzyme, comprising the following steps: A: adding soil to a precooled ion remover and a phosphate buffer in sequence; evenly blending, carrying out centrifugation on the mixture at room temperature and collecting sediment; B: adding the sediment obtained in the step A to a cell sample protein extracting solution, carrying out vortex blending, breaking cells by using an ultrasonic method in ice-bath, carrying out centrifugation to collect supernate, adding solid ammonium sulphate until the solubility is reached, oscillating while blending, carrying out centrifugation to collect precipitated protein, adding a dissolved solution to dissolve the protein, and preserving the dissolved protein; and C: placing 1,5-ribulose diphosphate carboxylase/oxygenase protein solution obtained in the step B in water bath to recover enzymatic activity and using an ultraviolet spectrophotometer to measure the activity of 1,5-ribulose diphosphate carboxylase/ oxygenase and the nanomole enzymatic activity of fixed CO2 in each kilogram of the soil. The simple, convenient and fast method has the advantage of good repeatability of measurement results and suitability for extraction and activity measurement of soil mircoorganism carbon-sequestrated key enzyme 1,5-ribulose diphosphate carboxylase/oxygenase.

Description

technical field [0001] The invention relates to the fields of biochemistry and environment, and more specifically relates to a method for extracting and measuring soil 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO). Background technique [0002] my country is facing great pressure to reduce greenhouse gas emissions, and enhancing the carbon sink function of terrestrial ecosystems is one of the important mechanisms for reducing greenhouse gas emissions. CO 2 The biological fixation is mainly carried out by plants and autotrophic microorganisms. Among them, autotrophic microorganisms are widely distributed in different ecosystems. They have strong environmental adaptability and can participate in CO2 under various environmental conditions, including craters, deep oceans, lake basins and other habitats where plants cannot survive. 2 fixed. Therefore, from the perspective of material and energy flow in the entire biosphere, it is of the most practical significance ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12Q1/527G01N21/33
Inventor 葛体达袁红朝吴金水肖和艾刘守龙童成立
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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