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Analysis method for detecting cellulase activity of biomembranes

An analysis method, cellulase technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that affect the contrast of the experiment, do not mention the pretreatment method, and it is difficult to ensure the consistency, etc., to achieve Ease of operation, simple method, and shortened reaction time

Active Publication Date: 2010-12-08
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The substrate used in measuring the total enzyme activity in the paper is filter paper, and the filter paper itself is not easy to disperse in the solution. The reliability of the method's measurement results is affected by several factors, such as the weight of the substrate per unit area size (each 1*6cm). Changes, cutting methods of filter paper, etc., it is difficult to ensure the consistency of each operation, which affects the contrast of the experiment
More importantly, this method is mainly aimed at the determination of the activity of cellulase preparations, and does not mention the pretreatment methods of other biological samples.

Method used

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  • Analysis method for detecting cellulase activity of biomembranes
  • Analysis method for detecting cellulase activity of biomembranes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The sample used in this example is a biofilm cultured in an aerobic bioreactor with cellobiose as a carbon source for 96 hours, with microcrystalline cellulose as a substrate, and using the above method to detect cellulase activity at different reaction times , to verify whether it is reasonable to choose 0.5h as the reaction time for this method.

[0029] step 1

[0030] With the glucose solution of known concentration as the standard solution, detect its absorbance with a spectrophotometer, make a standard curve with the glucose concentration C of the standard solution and the absorbance value A measured, and obtain the relational formula A=0.0347+0.70118 C;

[0031] step 2

[0032] Get the biofilm and place it in a beaker, and break it with an ultrasonic cell breaker at a frequency of 20KC for 5 minutes, and get 100ml of the bacterial suspension therein and dry it to a constant weight filter paper (weight is m 0 ) and filtered, put into an oven at 103°C for drying,...

Embodiment 2

[0043] The sample used in this example is a biofilm cultured for 96 hours in a eutrophic bioreactor with cellobiose as a carbon source, using filter paper and microcrystalline cellulose as substrates, and using the corresponding FPA method and the above method to detect the same Cellulase activity of samples.

[0044] step 1

[0045] With the glucose solution of known concentration as the standard solution, detect its absorbance with a spectrophotometer, make a standard curve with the glucose concentration C of the standard solution and the absorbance value A measured, and obtain the relational formula A=0.0347+0.70118 C;

[0046] step 2

[0047] Get the biofilm and place it in a beaker, break it with an ultrasonic cell breaker, frequency 20KC, break it for 10 minutes, get 100ml of bacterial suspension therein and dry it to constant weight filter paper (weight is m 0 ) and filter, put it into an oven at 105°C for drying, take it out and weigh it after cooling in a desiccator...

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Abstract

The invention discloses an analysis method for detecting the cellulase activity of biomembranes. The method specifically comprises the following steps of: (1) taking a proper quantity of cultured biomembranes, putting the biomembranes in a beaker, disrupting by using an ultrasonic cell disruption instrument,wherein the frequency is 20KC, the disrupting time is 5-10 minutes; and taking 100ml of bacterial suspension for measuring MLSS (Mixed Liquor Suspended Solids); (2) centrifuging the remained bacterial suspension, taking the supernate and adding a phosphate buffer and substrate microcrystalline cellulose for reacting for 30 minutes at constant temperature; (3) immediately adding a DNS (3,5-Dinitrosalicylic acid) reagent for terminating the reaction after the reaction ends, and putting the beaker in a boiling water bath for heating for 5 minutes; taking out for obtaining a constant volume, uniformly shaking, measuring the absorbance by using a spectrophotometer, and doing a blank experiment meanwhile; and (4) calculating the cellulase activity. The invention shortens the reaction time, has simple method and easy operation and simplifies a calculation process.

Description

technical field [0001] The invention relates to the measurement of cellulase activity, in particular to an analysis method for detecting cellulase activity of biofilm. Background technique [0002] As a method of treating wastewater, the biofilm method has developed quite maturely and has been widely used. This method relies on the enzymes produced by microorganisms to catalyze the degradation of pollutants, so that the water quality can be purified. In the process of industrial wastewater treatment, the cellulase produced by microorganisms is directly related to the degradation rate of macromolecular organic matter such as cellulose, hemicellulose, and lignin in wastewater. Cellulase activity has become an important parameter for evaluating the operation effect of treatment facilities. Therefore, it is of great significance to establish a relatively fast and accurate detection method for biofilm cellulase activity. [0003] The determination of cellulase activity basicall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34
Inventor 万金泉黄蓉姿王艳马邕文郭文杰
Owner SOUTH CHINA UNIV OF TECH
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