Method for measuring activity of cellobiohydrolase I in subtropical forest soil

A technology of cellobiose and forest soil, which is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of shortening the measurement time and difficult measurement, and achieve the effect of improving accuracy and efficiency and saving costs

Inactive Publication Date: 2015-10-21
WEIFANG MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

This assay method uses p-nitrophenyl cellobioside as the specific substrate of cellobiohydrolase Ⅰ, which solves the problem of difficult determination of single cellulase activity, and the enzymatic hydrolysis reaction is carried out under the optimal conditions. Using a multi-functional microplate reader improves the accuracy, shortens the measurement time and saves the cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] A method for measuring cellobiohydrolase I activity in subtropical forest soil consists of the following steps:

[0021] (1) p-nitrophenyl cellobioside was prepared into a substrate solution with a concentration of 1.2 mM with 50 mM sodium acetate buffer solution, the pH of which was 5.0;

[0022] (2) Take 160 μL of the substrate solution prepared in step (1) into the EP tube, weigh 0.06g of soil and add it into it, mix well and place it in a water bath at a constant temperature of 40°C, and react for 90 minutes; after the reaction, add 0.1mL 0.5M NaCO 3 The solution terminates the reaction;

[0023] (3) The solution after the reaction in step (2) is placed in a centrifuge and centrifuged at 5000r / min for 5min;

[0024] (4) Take 100 μL of the supernatant from step (3), and use a multi-functional microplate reader to measure the light absorption value at OD400nm;

[0025] (5) The activity of cellobiohydrolase Ⅰ in subtropical forest soil was estimated by the standard ...

Embodiment 2

[0029] A method for measuring cellobiohydrolase I activity in subtropical forest soil consists of the following steps:

[0030] (1) p-nitrophenyl cellobioside was prepared into a substrate solution with a concentration of 1.0 mM with 40 mM sodium acetate buffer solution, the pH of which was 4.5;

[0031] (2) Take 140 μL of the substrate solution prepared in step (1) into an EP tube, weigh 0.04 g of soil and add it into it, mix well, place in a water bath at a constant temperature of 45°C, and react for 60 minutes; after the reaction, add 0.1 mL 0.4M NaCO 3 The solution terminates the reaction;

[0032] (3) The solution after the reaction in step (2) is placed in a centrifuge and centrifuged at 4000r / min for 8min;

[0033] (4) Take 100 μL of the supernatant from step (3), and use a multi-functional microplate reader to measure the light absorption value at OD400nm;

[0034] (5) The activity of cellobiohydrolase Ⅰ in subtropical forest soil was estimated by the standard curve...

Embodiment 3

[0038] A method for measuring cellobiohydrolase I activity in subtropical forest soil consists of the following steps:

[0039] (1) p-nitrophenyl cellobioside was prepared into a substrate solution with a concentration of 1.5 mM with 60 mM sodium acetate buffer solution, the pH of which was 4.8;

[0040] (2) Take 150 μL of the substrate solution prepared in step (1) into an EP tube, weigh 0.05 g of soil and add it, mix well, place in a water bath at a constant temperature of 50°C, and react for 80 minutes; after the reaction, add 0.1 mL 0.5M NaCO 3 The solution terminates the reaction;

[0041] (3) The solution after the reaction in step (2) is placed in a centrifuge and centrifuged at 4500r / min for 7min;

[0042] (4) Take 100 μL of the supernatant from step (3), and measure the light absorption value at OD420nm with a multi-functional microplate reader;

[0043] (5) The activity of cellobiohydrolase Ⅰ in subtropical forest soil was estimated by the standard curve.

[0044...

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Abstract

The invention relates to a method for measuring the activity of cellobiohydrolase I in the subtropical forest soil, and especially relates to a method utilizing a multifunctional enzyme-linked immunosorbent assay (ELISA) instrument to measure the activity of cellobiohydrolase I in the subtropical forest soil. According to the provided method, nitrobenzene cellobioside is taken as the substrate, then the substrate is mixed with a soil sample for reactions in a water bath, and after the reactions, a multifunctional ELISA instrument is used to measure the reaction products. The provided measuring method has the advantages of simpleness, efficiency, and accuracy.

Description

Technical field [0001] The present invention involves a method of determining the activity of fiber diet enzyme Ⅰ activity in the subtropical forest soil, especially a method that uses a multi -functional enzyme label to determine the active of fiber diet enzyme Ⅰ in the subtropical forest soil. Background technique [0002] Cellulose is one of the important components of plant residues entering the soil. Among soil organic components, cellulose accounts for about 40%-55%of plant organic matter, and together with lignin, it accounts for 60-75%of soil organic matterEssenceCellulose is a macromolecular polysaccharide composed of β-1 and 4 glycoside bonds of glucose, and the biological conversion process is completed under the action of fibinase.Celloinase is a type of compound enzyme, including three different types of complexases: outer glucosacidase, internal glucosacidase, and β-glucosidase.Among them, fibromanohease Ⅰ is an important outer glucosacidase, which can decompose cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34
Inventor 吕艳娜李文静武翔宇
Owner WEIFANG MEDICAL UNIV
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