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A method for measuring the activity of acetolactate synthase by using acetolactate decarboxylase to catalyze decarboxylation and its application

A technology of acetolactate decarboxylase and acetolactate, which is applied in the field of biological enzymology detection, can solve problems such as not seen, and achieve the effect of high efficiency

Inactive Publication Date: 2019-02-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method can reflect the existence of acetolactate synthase, but there are still some limitations in the accurate determination of the activity of the enzyme, and this method is not suitable for the detection of other types of biological materials such as fungi and bacterial acetolactate synthase activity, so it is not applicable. See reports of its application to materials other than canola

Method used

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  • A method for measuring the activity of acetolactate synthase by using acetolactate decarboxylase to catalyze decarboxylation and its application
  • A method for measuring the activity of acetolactate synthase by using acetolactate decarboxylase to catalyze decarboxylation and its application
  • A method for measuring the activity of acetolactate synthase by using acetolactate decarboxylase to catalyze decarboxylation and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Rapeseed rape leaves are used as representatives of higher plant dicotyledonous green tissue materials. Take 3-5g of leaves of rapeseed (the variety is Haoyou 11), add liquid nitrogen and grind it into powder, add enzyme extraction buffer according to the ratio of W / V (leaf fresh weight g: buffer volume ml) = 1:5, and mix well After 20 minutes in ice bath, filter with two layers of gauze, centrifuge the filtrate at 4°C, 12000 rpm for 20 minutes, and obtain the supernatant as crude enzyme solution.

[0072] Then take the crude enzyme solution and measure the activity of acetolactate synthase according to the above-mentioned steps of measuring the enzyme activity. Measure 3 parallel tubes, repeat 3 times, the reaction solution shows red, and the color development of the reaction solution of 3 parallel tubes in one measurement is shown in figure 1 .

[0073] Randomly take a tube of reaction solution and scan the absorbance in the range of 400-620nm. The results show that...

Embodiment 2

[0076] Rice leaves are used as representatives of higher plant monocot green tissue materials. Take 6-10g of rice leaves (the variety is TN1), add liquid nitrogen and grind them into powder, add enzyme extraction buffer according to the ratio of W / V (leaf fresh weight g:buffer volume ml)=1:5, mix well, After cooling in ice for 20 minutes, filter with two layers of gauze, centrifuge the filtrate at 4° C., 12000 rpm for 20 minutes, and obtain the supernatant as crude enzyme solution.

[0077] Then take the crude enzyme solution and measure the activity of acetolactate synthase according to the above-mentioned steps of measuring the enzyme activity. The measurement experiment was repeated 3 times. Measure 3 parallel tubes, repeat 3 times, you can see that the reaction solution is red, and the color of the reaction solution of 3 parallel tubes in one measurement is shown in figure 1 . Randomly take a tube of reaction solution and scan the absorbance in the range of 400-620nm. T...

Embodiment 3

[0080] The root of rice is used as a representative of non-green tissue materials of higher plants. Take the root of rice (the variety is TN1), clean it with distilled water, absorb the surface moisture with absorbent paper, weigh 5-10g of material, add liquid nitrogen and grind it into powder, press W / V (root fresh weight g: buffer volume ml) = 1:5 ratio of enzyme extraction buffer was added, mixed well, ice-bathed for 20 minutes, filtered with two layers of gauze, and the filtrate was centrifuged at 4°C, 12000rpm for 20 minutes, and the obtained supernatant was the crude enzyme solution.

[0081] Then take the crude enzyme solution and measure the activity of acetolactate synthase according to the above-mentioned steps of measuring the enzyme activity. The measurement experiment was repeated 3 times, and the reaction solution showed red color. figure 1 . Randomly take a tube of reaction solution and scan the absorbance in the range of 400-620nm. The results show that the r...

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Abstract

The invention discloses a method for testing activity of acetolactate synthetase through catalyzing and decarboxylation by acetolacetate decearboxylase. The method comprises the following steps that (1) coarse acetolactate synthetase solution, acetolacetate decearboxylase, distilled water and sodium-pyruvate-containing reaction buffer solution are uniformly mixed; then, enzymatic reaction is performed to obtain enzymatic reaction liquid; (2) a color developing agent is added into the enzymatic reaction liquid and blank control liquid for color developing reaction; supernatant fluid is taken through centrifugation after the color development; the light absorption degree A<530> in a 530nm wavelength position of the enzymatic reaction liquid is detected by a spectral photometer; the enzyme activity of the coarse acetolactate synthetase solution is calculated according to the following formula of the activity of the acetolactate synthetase equals to delta A<530> / ([Pr].t). The method can be widely used for testing the activity of the acetolactate synthetase in various biological materials such as plants, fungi and germs, and can be used for screening or identifying acetolactate synthetase inhibitors.

Description

technical field [0001] The invention relates to a new method for measuring the activity of acetolactate synthetase by enzymatically catalyzing decarboxylation, which is a bioenzyme detection technology and belongs to the field of biotechnology. Background technique [0002] Acetolactate synthase (acetolactate synthase, EC 4.1.3.18), also known as acetohydroxyacid synthase (acetohydroxyacid synthase), is a branched-chain amino acid (leucine, isoleucine, valine, etc.) biological Key enzyme in the first stage of the synthesis process. By inhibiting the activity of acetolactate synthase, the biosynthesis of branched-chain amino acids in plants can be destroyed, protein synthesis is hindered, thereby interfering with cell division, affecting plant growth, and causing plant death. Because animals (including humans) do not contain acetolactate synthase, this enzyme has always been an important target in the development of herbicides. Acetolactate synthase inhibitor herbicides hav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/527
CPCC12Q1/527
Inventor 梁五生温雪玮武斌陈泱泱娄永根
Owner ZHEJIANG UNIV
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