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Method for rapidly detecting activity of theanine and glutamine hydrolase in tea trees

A technology of glutamine and theanine, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of inseparability, high cost, low work efficiency, etc., and achieves the elimination of background interference, the method is simple, the speed quick effect

Inactive Publication Date: 2016-01-27
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the HPLC method has the following disadvantages: 1. The method is cumbersome, requiring extraction, separation, purification, concentration and derivation of the reaction product; 2. The cost is high, requiring expensive equipment and derivative reagents, including HPLC systems, fluorescence detectors, 2,4- Dinitrofluorobenzene (DNFB), o-phthalaldehyde (OPT), etc.; 3. It is toxic to the human body, and the reagents used include DNFB, OPT, ethyl acetate, methanol and acetonitrile, etc., which are toxic to the human body, and are easy to pollute the environment. ; 4. The analysis time is long, and it takes 1-2 hours to detect a sample by HPLC method, the work efficiency is not high, and it cannot be applied to large-scale screening analysis
However, theanine, glutamine and glutamic acid are relatively similar in nature and structure, and the paper chromatography method known at present that can realize large-scale analysis cannot completely separate the three, so it cannot be used for theanine Activity detection of acid hydrolase and glutamine hydrolase

Method used

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  • Method for rapidly detecting activity of theanine and glutamine hydrolase in tea trees
  • Method for rapidly detecting activity of theanine and glutamine hydrolase in tea trees
  • Method for rapidly detecting activity of theanine and glutamine hydrolase in tea trees

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Embodiment 1

[0042] Embodiment 1 measures the method for theanine hydrolase enzyme activity in tea tree

[0043] This embodiment provides a method for measuring the activity of theanine hydrolase in tea tree, including:

[0044] Main equipment: 1. Thin-layer electric spotting instrument; 2. UV-Vis spectrophotometer;

[0045] 3. Constant temperature water bath; 4. Centrifuge; 5. pH meter.

[0046] Materials and Reagents

[0047] 1. Materials: fresh leaves of tea tree, stored at -80°C for later use.

[0048] 2. Main reagents:

[0049] (1) Polyvinylpyrrolidone (PVPP): It is used to adsorb polyphenols in the crude enzyme solution and maintain a high activity of the enzyme protein.

[0050] (2) 1MpH7.0 potassium phosphate buffer configuration:

[0051] Stock solution A: Weigh 114.11 g of dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O), dissolve it in 500 mL of distilled water, and set aside.

[0052] Stock solution B: Weigh 68.05g of potassium dihydrogen phosphate (KH2PO4), dissol...

Embodiment 2

[0081] Embodiment 2 measures the method for glutamine hydrolase enzyme activity in tea tree

[0082] The present embodiment provides a method for measuring glutamine hydrolase enzyme activity in tea tree, comprising:

[0083] Main equipment: same as embodiment 1

[0084] Materials and Reagents:

[0085] 1. Material: Same as Example 1

[0086] 2. Main reagents:

[0087] (1) 70mM glutamine: Weigh 1023.05mg glutamine, dissolve it in 100mL distilled water, and set aside.

[0088] All the other reagents are the same as in Example 1

[0089] Operation method:

[0090] (1) crude enzyme liquid preparation: with embodiment 1

[0091] (2) Detection of protein content: same as in Example 1, the protein content (P) was 1.446 mg.

[0092] (3) Enzyme activity detection

[0093] ① A portion of crude enzyme solution was added to glutamine solution to undergo hydrolysis reaction. Glutamine hydrolase reaction system (1mL) was shown in Table 1, 32°C water bath, 60min, immediately after t...

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Abstract

The invention relates to a method for rapidly detecting activity of theanine and glutamine hydrolase in tea trees. The method includes the steps of firstly, preparing crude enzyme; secondly, detecting the content P of protein in crude enzyme; thirdly, adding crude enzyme to a theanine solution or a glutamine solution to obtain hydrolysis reaction system samples, conducting centrifuging after the reaction is completed, and taking supernate; fourthly, detecting the light absorption value of the hydrolysis reaction product glutamic acid in supernate through high performance thin layer chromatography, substituting the light absorption value into a linear equation, calculating the concentration N of glutamic acid, and then calculating the content M of glutamic acid and the activity of theanine hydrolase or glutamine hydrolase. The method has the advantages of being high in flux and efficiency, and a solid experimental technological foundation is laid for research on metabolism of theanine in tea trees, separation and purification of theanine hydrolase and glutamine hydrolase, gene cloning and function verification of zymoprotein.

Description

technical field [0001] The invention belongs to the technical field of biological material detection, and in particular relates to a method for detecting enzyme activities of theanine hydrolase and glutamine hydrolase in tea trees. Background technique [0002] Theanine (N-ethyl-γ-L-glutamine) is a characteristic non-protein amino acid in tea tree. At present, it has only been reported in Qin (Xerocomusbadius) and some camellia plants (Camellia japonica and Camellias asanqua). Theanine accounts for about 70% of the total amount of free amino acids in tea shoots and leaves, and is an important factor affecting the quality of green tea. At the same time, theanine has important physiological activities and pharmacological functions such as calming the nerves, anti-cancer, and anti-depression. [0003] In the process of theanine metabolism, theanine synthase and theanine hydrolase directly participate in the synthesis and decomposition of theanine, alanine transaminase, alanin...

Claims

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Application Information

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IPC IPC(8): G01N30/90
Inventor 李勤刘仲华黄建安林海燕张振乾
Owner HUNAN AGRICULTURAL UNIV
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