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Method for visually determining SOD enzyme activity

An enzymatically active and intuitive technology that can be used in the biological field to solve problems such as cumbersome operations

Pending Publication Date: 2019-11-22
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are all indirectly measuring the SOD enzyme activity after the enzyme is extracted, and the operation is cumbersome.

Method used

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  • Method for visually determining SOD enzyme activity
  • Method for visually determining SOD enzyme activity
  • Method for visually determining SOD enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Using wild-type Bacillus cereus 0-9, mutant ΔSOD A1 (wild-type knockout of ΔsodA1) and mutant FSDS (wild-type knockout of sodA1, sodA2, sodC, sodS four SOD enzyme genes) as objects, respectively measure SOD Enzyme activity, the specific operation is as follows:

[0026] Liquid medium composition: LB medium, natural pH, sterilized at 121°C for 20min.

[0027] Solid medium composition: LB medium, natural pH, 1% agar, sterilized at 121°C for 250min, adding autoclaved or filter-sterilized sodium selenite at 50°C to make selenite in the solid medium The concentration of sodium is 2mM. Shake well and pour it into a plate. After solidification, seal it and store it for later use.

[0028] The bacterial strain was first activated into a liquid bacterial suspension, and then inoculated into the seed medium at an inoculation amount of 5ml / 100ml, cultivated at 30°C for 24h, and the shaking table rotated at a speed of 180r / min. Then, insert the cultured seeds into the liquid ferm...

Embodiment 2

[0030] Using wild-type Bacillus cereus 0-9, mutant ΔSOD A1 (wild-type knockout of ΔsodA1) and mutant FSDS (wild-type knockout of sodA1, sodA2, sodC, sodS four SOD enzyme genes) as objects, respectively measure SOD Enzyme activity, the specific operation is as follows:

[0031] Liquid medium composition: LB medium, natural pH, sterilized at 121°C for 20min.

[0032] Composition of solid medium: LB medium, natural pH, 1% agar, sterilized at 121°C for 250 minutes, added filtered paraquat at 90°C to make the concentration of paraquat in the solid medium 2mM, shake well Finally, pour it into a flat plate, seal it and store it for later use after solidification.

[0033] The bacterial strain was first activated into a liquid bacterial suspension, and then inoculated into the seed medium at an inoculation amount of 10ml / 100ml, cultivated at 30°C for 24h, and the shaking table rotated at a speed of 180r / min. Then, insert the cultivated seeds into the liquid fermentation medium with ...

Embodiment 3

[0035] Using wild-type Bacillus cereus 0-9, mutant ΔSOD A1 (wild-type knockout of ΔsodA1) and mutant FSDS (wild-type knockout of sodA1, sodA2, sodC, sodS four SOD enzyme genes) as objects, respectively measure SOD Enzyme activity, the specific operation is as follows:

[0036] Liquid medium composition: LB medium, natural pH, sterilized at 121°C for 20min.

[0037] Solid medium composition: LB medium, natural pH, 1% agar, sterilized at 121°C for 250min, added vitamin K after filter sterilization at 50-90°C 3 , so that vitamin K in solid medium 3 The concentration of the solution is 5mM, shake it well, pour it into a plate, and seal it for storage after solidification.

[0038] The bacterial strain was first activated into a liquid bacterial suspension, and then inoculated into the seed medium at an inoculum amount of 1ml / 100ml, cultivated at 30°C for 24h, and the shaking table rotated at 180r / min. Then, insert the cultivated seeds into the liquid fermentation medium according...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for visually determining the SOD enzyme activity. The method comprises the steps of inoculating a strain into a liquid culture medium to culture a fermentation liquid, carrying out plate coating on a solid culture medium containing a substance capable of stimulating cells to generate free radicals, culturing the strain to an exponential growth phase after plate coating, and observing the size of a bacterial colony, wherein the smaller the bacterial colony is, the smaller the SOD enzyme activity is. Through the method, the SOD enzyme activity can be visually measured, the cells do not need to be crushed to extract an enzyme solution, and the operation is simple.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for visually measuring SOD enzyme activity. Background technique [0002] Superoxide radicals (O 2 · - ) is a typical reactive oxygen species (ROS), generally considered to be the precursor of other ROS, ROS can cause oxidative stress and oxidative damage, thereby causing cell senescence and apoptosis. Peroxidase (SOD, EC1.15.1.1) is the first enzyme found to have a breakthrough effect on scavenging free radicals. This enzyme can catalyze the following reactions: [0003] o 2 · - +O 2 · - +2H + →O 2 +H 2 o 2 [0004] There are many methods for measuring SOD enzyme activity in the prior art, such as non-denaturing polyacrylamine gel separation combined with NBT staining (Leonowicz, PLoS One 2018), automatic oxidation analysis of pyrogallol, cytochrome C reduction Analytical methods, etc. (Attar, 2006, Appl. Biochem. Microbiol. 2006). However, these m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/28C12Q1/02C12R1/085
CPCC12Q1/02C12Q1/28G01N2333/90283
Inventor 马云峰王刚刘晴
Owner HENAN UNIVERSITY