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Method for measuring activity of beta-glucan synthase

A technology for glucan synthase and activity, which is applied in the field of determining the activity of beta-glucan synthase, can solve the problems of harm, high experimental cost, expensive substrates and the like, and achieves simple and safe operation, high sensitivity and high safety. Effect

Inactive Publication Date: 2012-05-09
HENAN UNIVERSITY OF TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the detection methods of the enzyme activity are based on UDP-[ 14 C] Glucose as the substrate, UDP-[ 14 C] The change of the radioactivity of glucose is used to determine the activity of β-glucan synthase, which requires expensive substrates (UDP-[ 14 C] glucose) and detection instrument, the experiment cost is high, and the radioactive substrate UDP-[ 14 C] Glucose is obviously harmful, so this kind of method is difficult to be widely used

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Main instruments: digital display electric heating constant temperature water bath, HH-4 type, Henan Zhicheng Technology Development Co., Ltd.; portable waterproof pH / mV / ℃ tester, HI8424 type, HANNA instruments; visible spectrophotometer, type 723, Shanghai Precision Scientific Instruments Ltd.

[0031] (1) Sample preparation—preparation of β-glucan synthase solution

[0032] Accurately weigh 1.0g β-glucan synthase, add 50ml phosphate buffer (7.0mmol / LNaH 2 PO 4 , 7.0mmol / L Na 2 HPO 4 , pH=6.8) stirred to dissolve, the final concentration of the sample was 0.02g / ml, and stored at 4°C for later use;

[0033] (2) Preparation of glucose standard solution

[0034] Accurately weigh 100mg of anhydrous glucose, add distilled water to make up to 100ml, and obtain a glucose standard solution with a concentration of 1.0mg / ml;

[0035] (3) Preparation of DNS solution

[0036] Weigh 3.15g of 3,5-dinitrosalicylic acid (AR), add 500ml of water, stir to dissolve, and take a wat...

Embodiment 2

[0051] Main instruments: digital display electric heating constant temperature water bath, HH-4 type, Henan Zhicheng Technology Development Co., Ltd.; portable waterproof pH / mV / ℃ tester, HI8424 type, HANNA instruments; visible spectrophotometer, type 723, Shanghai Precision Scientific Instruments Ltd.

[0052] (1) Sample preparation—preparation of β-glucan synthase solution

[0053] Accurately weigh 2.0g β-glucan synthase, add 50ml phosphate buffer (7.0mmol / LNaH 2 PO 4 , 7.0mmol / L Na 2 HPO 4 , pH=6.8) stirred to dissolve, the final concentration of the sample was 0.04g / ml, and stored at 4°C for later use;

[0054] (2) Preparation of glucose standard solution

[0055] Accurately weigh 100mg of anhydrous glucose, add distilled water to make up to 100ml, and obtain a glucose standard solution with a concentration of 1.0mg / ml;

[0056] (3) Preparation of DNS solution

[0057] Weigh 3.15g of 3,5-dinitrosalicylic acid (AR), add 500ml of water, stir to dissolve, and take a wat...

Embodiment 3

[0072] Main instruments: digital display electric heating constant temperature water bath, HH-4 type, Henan Zhicheng Technology Development Co., Ltd.; portable waterproof pH / mV / ℃ tester, HI8424 type, HANNA instruments; visible spectrophotometer, type 723, Shanghai Precision Scientific Instruments Ltd.

[0073] (1) Sample preparation—preparation of β-glucan synthase solution

[0074] Accurately weigh 5.0g β-glucan synthase, add 50ml phosphate buffer (7.0mmol / LNaH 2 PO 4 , 7.0mmol / L Na 2 HPO 4 , pH=6.8) stirred to dissolve, the final concentration of the sample was 0.1g / ml, and stored at 4°C for later use;

[0075] (2) Preparation of glucose standard solution

[0076] Accurately weigh 100mg of anhydrous glucose, add distilled water to make up to 100ml, and obtain a glucose standard solution with a concentration of 1.0mg / ml;

[0077] (3) Preparation of DNS solution

[0078] Weigh 3.15g of 3,5-dinitrosalicylic acid (AR), add 500ml of water, stir to dissolve, and take a wate...

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Abstract

The invention discloses a method for measuring the activity of beta-glucan synthase. The method disclosed by the invention has the advantage that the activity of the beta-glucan synthase is determined by detecting the amount of glucan consumed by synthesizing the beta-glucan synthase into beta-glucan with the glucan as an enzymatic reaction substrate and 3,5-dinitrosalicylic acid as a color developing agent under visible light. Compared with a traditional method for measuring the activity of the beta-glucan synthase by measuring the change of radioactive activity of UDP (Uridine Diphosphate)-[14C] glucan through a liquid scintillation counting instrument by using the UDP-[14C] glucan as a substrate, and the experiment cost is greatly reduced. In addition, no radioactive glucan is used as the substrate in the method, thus the safety of the experiment is high. The method has the advantages of high sensitivity, simpleness and safety in operation, convenience, quickness and convenience for large-scale popularization and application. The method disclosed by the invention can be widely used for researching and developing saccharide and enzyme products.

Description

technical field [0001] The invention relates to the field of enzymology and enzyme technology, in particular to a method for measuring the activity of β-glucan synthase. Background technique [0002] β-glucan is a kind of non-starch polysaccharide, which is composed of β-1,3-glucosidic bonds and / or β-1,6-glucosidic bonds and a small amount of β-1,4-glucosidic bonds The chain polysaccharide is an important component of fungal and plant cell walls. A large number of chemical and pharmacological analyzes have shown that fungal polysaccharides rich in β-glucan (such as Grifola frondosa polysaccharide, Lentinan) have obvious anti-tumor, anti-virus, immune regulation, blood sugar regulation, blood pressure lowering and other effects. β-glucan synthase plays a vital role in the synthesis of β-glucan. At present, most of the detection methods of the enzyme activity are based on UDP-[ 14 C] Glucose as the substrate, UDP-[ 14 C] The change of the radioactivity of glucose is used t...

Claims

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Application Information

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IPC IPC(8): C12Q1/48G01N21/31
Inventor 王卫国李鹏赵永亮侯启昌朱奎成崔羽佳古亚楠孟甜甜张鹏丁金聚
Owner HENAN UNIVERSITY OF TECHNOLOGY
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