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Ferulaic acid esterase activity detection method

A ferulic acid esterase and activity detection technology, applied in the field of enzyme engineering, can solve the problems of expensive equipment, time-consuming and laborious, complicated operation, etc., and achieve the effects of low sample consumption, high application value, and accurate experimental results

Inactive Publication Date: 2010-06-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the equipment required by HPLC and GC is expensive, the operation is complicated, and the cost is high. The relatively low cost of using a spectrophotometer for detection is relatively low, but these three methods require a large amount of samples, and the steps are cumbersome, time-consuming and labor-intensive.

Method used

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  • Ferulaic acid esterase activity detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0033] The ferulic acid esterase activity detection method of the present invention is used to measure the daily change of the ferulic acid esterase activity in the rumen fluid of beef cattle.

[0034] (1) Weigh an appropriate amount of 104.11mg of methyl ferulate, fully dissolve it in 20ml of absolute ethanol, dilute it to 100μM with 100mM MOPS buffer (pH6.0), and preheat at 39°C for 15 minutes to obtain methyl ferulate solution;

[0035] (2) Add 100 μL of the enzyme solution to be tested diluted 6.25 times to each well of the 96-well plate, and preheat at 39°C for 15 minutes;

[0036] (3) Then, in the enzyme solution to be detected in step (2), add 200 μL of methyl ferulate solution in each of the 48 wells as a reaction system, and react at 39° C. for 30 minutes;

[0037] (4) Use a microplate reader to detect the OD value of the reaction system at the beginning and end of the reaction at 340nm;

[0038] 100mM MOPS buffer (pH6.0) was used to replace the methyl ferulate solu...

Embodiment 2

[0046] Utilize ferulic esterase activity detection method of the present invention to measure ferulic esterase activity in beef cattle rumen juice, specifically comprise the steps:

[0047] (1) Weigh 104.11mg of methyl ferulate, fully dissolve in 20ml of absolute ethanol, dilute to 50μM with 100mM MOPS buffer (pH4.5), preheat at 35°C for 30 minutes to obtain methyl ferulate solution ;

[0048] (2) Add 50 μL of the enzyme solution to be tested appropriately diluted 6 times to each well of the 96-well plate, and preheat at 35°C for 30 minutes to obtain the enzyme solution to be tested;

[0049] (3) In the enzyme solution to be tested in step (2), add 100ul of methyl ferulate solution in step (1) to each of the 48 wells, react at 35°C for 30 minutes, and use a microplate reader to detect the reaction system at 340nm OD value at the beginning and end of the reaction;

[0050] Replace methyl ferulate solution with 100mM MOPS buffer (pH6.0) as the enzyme blank, and add 100mM MOPS ...

Embodiment 3

[0054] Utilize ferulic acid esterase activity detection method of the present invention to measure ferulic acid esterase activity in the ferulic acid esterase preparation (Biocatalysts Co.Ltd, UK), specifically comprise the steps:

[0055] (1) Weigh 104.11mg of methyl ferulate, fully dissolve in 20ml of absolute ethanol, dilute to 200μM with 100mM MOPS buffer (pH7), preheat at 65°C for 10 minutes to obtain methyl ferulate solution;

[0056] (2) Add 100 μL of the enzyme solution to be tested appropriately diluted 1600 times to each well of the 96-well plate, add a total of 8 wells, and preheat at 65°C for 10 minutes to obtain the enzyme solution to be tested;

[0057] (3) In the enzyme solution to be tested in step (2), add 200ul of methyl ferulate solution in step (1) to each of the four wells, react at 65°C for 10 minutes, and use a microplate reader to detect the reaction system at 340nm OD value at the beginning and end of the reaction;

[0058] Replace methyl ferulate solut...

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Abstract

The invention provides a method for detecting activity of ferulic acid esterase, which comprises the following steps of: mixing an enzyme solution to be detected with methyl ferulate solution to react, and calculating the enzyme activity. The invention can detect 12 samples on one plate with 96 holes with very small sample amount, thus simplifying the steps for detecting activity of ferulic acid esterase, greatly reducing the time, lowering working density and achieving accurate detection result and strong operability. The invention is suitable for batch activity detection of ferulic acid esterase.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a method for detecting ferulic esterase activity by measuring the OD value of a reaction system. Background technique [0002] Ferulic acid esterase (E.C.3.1.1.73, also known as cinnamic acid esterase, cinnamic acid hydrolase) is a class of enzymes that hydrolyze the ester bond between hydroxycinnamic acid and sugar in the plant cell wall, and the main reaction it catalyzes is : Polysaccharide ~ ferulic acid ester + H 2 O = ferulic acid + polysaccharides (including some derivatives of ferulic acid). [0003] Ferulic acid esterase has a wide application space in papermaking, food, pharmaceutical and feed industries. In recent years, the role of ferulic esterase in feed digestion has attracted increasing attention. About 30% to 80% of the dry matter of straw and forage grass is cell wall components, however, the digestion and utilization of these cell walls by rumin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31C12Q1/44G06F19/00
Inventor 杨红建岳群
Owner CHINA AGRI UNIV
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