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Method for detecting activity of benzyl alcohol acetyltransferase

A technology of acetyltransferase and benzyl alcohol, applied in the field of biochemistry, can solve the problems of unfavorable development and research of benzyl alcohol acetyltransferase, cumbersome operation steps, long experimental period and the like, and achieves elimination of influence, simple experimental operation steps, and detection results. Sensitive and accurate effects

Active Publication Date: 2018-04-20
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the operation steps of this method are cumbersome, the experimental period is long, and the cost is high, which is not conducive to the development and research of benzyl alcohol acetyltransferase in the biosynthetic pathway.

Method used

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  • Method for detecting activity of benzyl alcohol acetyltransferase
  • Method for detecting activity of benzyl alcohol acetyltransferase
  • Method for detecting activity of benzyl alcohol acetyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Analysis of Benzyl Alcohol Acetyltransferase Activity in Crude Extracted Protein of Plum Blossom Petals

[0027] This embodiment provides a method for detecting the activity of benzyl alcohol acetyltransferase in crude protein extracted from plum blossom petals, including:

[0028] 1. Mother liquor configuration of experimental reagents:

[0029] (1) Preparation of 10mM DTNB mother solution:

[0030] Weigh 0.2g of DTNB powder and dissolve it in 50mL 0.5M Tris-HCl (pH8.0), store in the dark, and prepare immediately after use;

[0031] (2) Preparation of 50mM acetyl-CoA mother solution:

[0032] Add 50mg of acetyl-CoA powder to 1.2mL of water, mix until complete dissolution, and store at -20°C;

[0033] (3) Preparation of 1M benzyl alcohol mother liquor:

[0034] Add 100 μL benzyl alcohol (analytical grade) to 864 μL DMSO, mix well, and store at -20°C;

[0035] (4) 1mM MgCl 2 Preparation of mother liquor:

[0036] 1M MgCl 2 Obtained by diluting the mothe...

Embodiment 2

[0058] Example 2 Analysis of Benzyl Alcohol Acetyltransferase Activity of Tobacco Leaf Crude Extract Protein

[0059] This embodiment provides a detection of the activity of benzyl alcohol acetyltransferase of the crude extract protein of tobacco leaves, including:

[0060] 1, experimental reagent mother solution configuration (same as embodiment 1)

[0061] 2. Configuration of protein benzyl alcohol acetyltransferase activity detection system:

[0062] (1) Experimental group:

[0063] Add 15 μL of DTNB mother solution, 15 μL of acetyl-CoA mother solution, 1.5 μL of benzyl alcohol mother solution, and MgCl in a 2 mL plastic centrifuge tube. 2 15 μL of the mother liquor and 200 μg of the crude tobacco leaf protein sample were made up to 1.5 mL with water, and mixed. The samples in the experimental group were repeated three times.

[0064] (2) Control group:

[0065] Boil 200 μg of crude tobacco leaf protein sample in boiling water for 10 minutes, centrifuge at 12,000 rpm f...

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Abstract

The invention relates to a method for detecting the activity of benzyl alcohol acetyltransferase. According to the method provided by the invention, a reaction mechanism that DTNB (5,5'-dithiobis-(2-nitrobenzoic acid) can have a mutual effect with sulfydryl to form colored thiophenol anions; the activity of the benzyl alcohol acetyltransferase in a protein sample is determined through detecting the generation amount of the sulfydryl in a reaction product. The method provided by the invention has the advantages that experiment operation steps are simple, the dosage of the sample is relatively less and a detection result is sensitive and accurate. When the method provided by the invention is applied, the efficient benzyl alcohol acetyltransferase can be relatively easily screened from different protein samples.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for detecting the activity of benzyl alcohol acetyltransferase in a protein sample to be tested in an in vitro enzyme reaction system. Background technique [0002] Benzyl acetate exists in the flowers and fruits of many plants, and its biosynthetic pathway uses benzyl alcohol and acetyl-CoA as substrates to generate benzyl acetate under the action of benzyl alcohol acetyltransferase. [0003] In the prior art, there is no relatively mature method for detecting the activity of benzyl alcohol acetyltransferase. In the research, mass spectrometry is generally used to characterize and quantify the reaction products, and then convert to obtain the parameters related to the enzyme activity. However, the operation steps of this method are cumbersome, the experiment period is long, and the cost is high, which is not conducive to the development and research of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 包菲张启翔程堂仁王佳
Owner BEIJING FORESTRY UNIVERSITY
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