Activity detection method of ester type catechin synthetase

A technology for detection of ester catechin and activity, which is applied in the directions of biochemical equipment and methods, determination/inspection of microorganisms, etc., can solve the problems such as the lack of a method for detecting the enzyme activity of ester catechin synthase, and achieves the improvement of tea High quality, avoid experimental operation, strong effect of accuracy

Inactive Publication Date: 2011-09-07
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

So far, there is no literature report on the enzyme activity detection method of ester-type catechin synthase

Method used

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  • Activity detection method of ester type catechin synthetase
  • Activity detection method of ester type catechin synthetase
  • Activity detection method of ester type catechin synthetase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Main equipment: 1. High performance liquid chromatography (HPLC); 2. Constant temperature water bath; 3. Rotary evaporator; 4. Centrifuge; 5. pH meter;

[0051] Materials and reagents:

[0052] 1. The tested material: fresh tea leaves (stored at -80℃ for later use);

[0053] 2. Main reagents

[0054] (1) Polyvinylpyrrolidone (PVPP), purchased from Shanghai Soleibao Biotechnology Co., Ltd., is used to adsorb polyphenols in the enzyme solution and maintain the higher activity of the enzyme protein;

[0055] (2) Quartz sand (commercially available): increase friction and increase the extraction rate of enzyme protein;

[0056] (3) 0.1 mol / L pH7.4 phosphate buffer

[0057] Solution A: Weigh out sodium dihydrogen phosphate (NaH 2 PO 4· 2H 2 O) 7.8 grams, dissolved in 500 mL of distilled water;

[0058] Solution B: Weigh disodium hydrogen phosphate (Na 2 HPO 4· 3H 2 O) 17.9 grams, dissolved in 500 mL of distilled water, ready for use;

[0059] Immediately before use, take a certain volum...

Embodiment 2

[0103] The main equipment is the same as in Example 1.

[0104] Materials and reagents:

[0105] 1. The tested material: the same as in Example 1;

[0106] 2. Main reagents:

[0107] Reaction substrate: 3.8 mmol / L epigallocatechin (EC) solution: Weigh 0.0011 g of EC, dissolve it in 1 mL of water, and store it below 4°C for later use. Other reagents are the same as in Example 1;

[0108] An ester type catechin synthase activity detection method includes the following steps:

[0109] 1.1. Preparation of enzyme solution

[0110] Same as in Example 1;

[0111] 1.2 Determination of enzyme activity

[0112] 1.2.1. Preparation of reaction solution

[0113] Mix 0.46mL enzyme solution, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EC) solution, 0.075mL reaction substrate 1-O-galloyl-β-D-glucose (βG) solution and 0.59mL phosphate buffer are mixed uniformly, and the temperature is 30℃ water bath for 60 minutes to obtain the reaction solution;

[0114] The...

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Abstract

The invention relates to an activity detection method of ester type catechin synthetase. The method comprises five steps, namely preparation of enzyme solution, preparation of reaction solution, preparation of contrast solution, detection of enzyme activity, and calculation of the enzyme activity. The ester type catechin serving as an enzyme reaction product is detected by using high performance liquid chromatography so as to detect the enzyme activity, and interference of oxidase and ester type catechin hydrolase on enzyme activity detection can be avoided by adding antioxidant and hydrolaseinhibitor in the reaction system can be avoided. The method is simple, has strong accuracy and high sensitivity, and can accurately detect the reaction product of the ester type catechin synthetase; and the operation is convenient, the detection time is short, fussy experiment operation is avoided, and the detection can be finished in 2 to 3 hours.

Description

technical field [0001] The invention belongs to the technical field of biological material detection, and in particular relates to a method for detecting the activity of ester-type catechin synthase. Background technique [0002] The ester type catechins in tea tree mainly include epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), and the non-ester type catechins mainly include epicatechin (EC) and epigallocatechin Catechin (EGC); Structurally, the ester catechins (epicatechin gallate (ECG) and epigallocatechin gallate (EGCG)) are in the The ester of gallic acid (GA) at the 3-OH position of the tea element (EC), epigallocatechin (EGC)) structure. [0003] The content of ester-type catechins in tea tree is relatively high, which is about 55% of the total amount of tea polyphenols, and is 12%-19% of the dry weight of fresh leaves [1] . Ester catechins have stronger medicinal value than non-ester catechins [2-3] , ester-type catechin has various pharmacological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/25
Inventor 高丽萍夏涛刘莉刘亚军
Owner ANHUI AGRICULTURAL UNIVERSITY
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