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Cellulase activity determination method based on micropore plate method

An assay method and micro-plate technology, applied in the field of biochemistry, can solve the problems of difficult large-scale promotion, expensive equipment and other problems, and achieve the effects of easy promotion, convenient operation and high precision

Inactive Publication Date: 2011-11-02
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this automated method can achieve rapid and high-throughput determination of enzyme activity, it requires relatively expensive equipment and is difficult to be widely used in actual production.

Method used

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  • Cellulase activity determination method based on micropore plate method
  • Cellulase activity determination method based on micropore plate method
  • Cellulase activity determination method based on micropore plate method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1) Establishment of standard curve

[0017] Take a 96-well PCR plate, add 40 μl of buffer solution and 20 μl of glucose solutions with concentrations of 0 mg / ml, 2.0 mg / ml, 3.3 mg / ml, 5.0 mg / ml, 6.7 mg / ml and 10 mg / ml in each well, and then Add 120 μl of DNS solution to each well, and incubate at 95° C. for 5 minutes in a gene amplification instrument. Finally, 40 μl was transferred from each well and placed in a microplate plate containing 160 μl water, and the absorbance was measured at 540 nm with a microplate reader.

[0018] Taking the glucose concentration as the abscissa and the absorbance as the ordinate, the regression equation Y=0.208X+0.0006 is obtained, and the standard curve is linear in the concentration range of 0mg / ml-10mg / ml, and the linear correlation coefficient is r=0.99975.

[0019] 2) Determination of filter paper enzyme activity in the sample

[0020] In a 96-well PCR plate, add 40 μl of pH4.8 sodium acetate buffer, 20 μl of 2-fold diluted enzym...

Embodiment 2

[0039] 1) Establishment of standard curve

[0040] The basic process is the same as in Example 1, after adding DNS solution and incubating at 90°C for 15 minutes, the regression equation is Y=0.203X+0.0008, the standard curve is linear in the concentration range of 0mg / ml-10mg / ml, and the linear correlation coefficient is r = 0.99935.

[0041] 2) Determination of endocellulase activity in the sample

[0042] The basic process is the same as in Example 1. The enzyme solution and substrate were incubated at 55°C for 30 minutes, then DNS solution was added, and incubated at 100°C for 5 minutes. The substrate used was 2% carboxymethylcellulose aqueous solution, and the endocellulose activity was as follows Formula (2) calculation.

[0043]

[0044] Formula (2)

[0045]Wherein IU / ml is the activity of filter paper enzyme in every milliliter of enzyme liquid; A 540 Sample is the enzymatic activity value that the sample records with the microporous plate DNS assay method; A 5...

Embodiment 3

[0060] 1) Establishment of standard curve

[0061] The basic process is the same as in Example 1, after adding DNS solution and incubating at 100°C for 5 minutes, the regression equation is Y=0.189X+0.0003, the standard curve is linear in the concentration range of 0mg / ml-10mg / ml, and the linear correlation coefficient is r = 0.99968.

[0062] 2) Determination of β-glucosidase activity in the sample

[0063] The basic process is the same as in Example 1. The enzyme solution and substrate are incubated at 45°C for 60 minutes, then DNS solution is added, and incubated at 90°C for 15 minutes. The substrate used is an aqueous solution of 0.5% salicylic acid, and the endocellulosic enzyme activity is according to the formula (2) calculation.

[0064] 3) Stability, precision and reproducibility experiments

[0065] The basic process is the same as in Example 1, and the results are shown in Table 7. The precision and repeatability are good, and the coefficients of variation of the...

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Abstract

A cellulase activity determination method based on a micropore plate method relates to the field of biochemistry, and specifically relates to a cellulase activity high flux determination method based on a micropore plate method. The method comprises steps of 1) establishing a glucose standard curve; 2) determining a sample enzyme activity; 3) checking out corresponding glucose content from the glucose standard curve according to an absorbance of the sample enzyme, and calculating an enzyme activity value according to a gram weight of generated glucose. The method is suitable for activity determination of filter paper enzyme, endo-cellulase, exo-cellulase and beta-glucosidase, and has characteristics of convenient operation, rapidness, simple equipment requirement and easiness for popularization, etc.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for high-throughput determination of cellulase activity based on a micro-orifice plate method. Background technique [0002] Cellulose is the primary product of photosynthesis and the most abundant renewable resource in the biosphere. With the improvement of living standards, organic wastes mainly composed of cellulose substances are increasing. Rational development and scientific utilization of this rich natural resource is of great significance to the sustainable development of human society. Using advanced technical means to transform it into energy, food and chemical raw materials that human beings urgently need is a key area of ​​research and development in countries all over the world. An effective way to completely decompose cellulose without pollution is to use the hydrolysis of cellulase. The key to using cellulase to hydrolyze cellulose into glucose is the product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 梁剑平李兆周王学红尚若峰郭文柱郭志廷郝宝成王曙阳陶蕾
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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