A group of amino acid sequences that can improve the stress resistance and stability of SOD in aqueous solution and its application

An amino acid and stress resistance technology, applied in biochemical equipment and methods, enzymes, oxidoreductases, etc., can solve problems such as short half-life and easy inactivation

Active Publication Date: 2017-04-12
北京迈恩泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Natural SOD has a short half-life in the process of industrial production and food and drug addition, and it is easily inactivated by denaturants and inhibitors.

Method used

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  • A group of amino acid sequences that can improve the stress resistance and stability of SOD in aqueous solution and its application
  • A group of amino acid sequences that can improve the stress resistance and stability of SOD in aqueous solution and its application
  • A group of amino acid sequences that can improve the stress resistance and stability of SOD in aqueous solution and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. Construct the complete sequence gene of Fe / Mn-SOD encoding NG80-2 ( sod-GTNG_2215 ), and construct the DNA sequence encoding the domain sodA of SOD-GTNG_2215 ( sodA-GTNG_2215 ), and determine the effects of inhibitors, detergents, and denaturants on the activity of expressed proteins.

[0064] 1.1 Extraction of total DNA from Bacillus thermodenitrophilus NG80-2 (CGMCC No. 1228)

[0065] In this example, the thermophilic denitrification bacillus NG80-2 ( Geobacillus thermodenitrificansThe strain has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and its preservation number is CGMCC No.1228). Take 3 mL of fresh culture cultured overnight, collect the bacteria by centrifugation, and suspend the bacteria in 250 μL of 50 mM Tris buffer (pH8 .0), add 10μL 0.4M EDTA (pH8.0), mix well and keep warm at 37°C for 20min, then add 30μL 20mg / L lysozyme, mix well and keep warm at 37°C for 20min, then add 5μL ...

Embodiment 2

[0124] build code B. subtilis Mn-SOD full sequence gene of BSn5 ( sod-BSn5 ) clone, and construct the recombinant SOD encoding ( SOD-GTNG_2215 The N-terminal sequence and B. subtilis The SODA recombination of BSn5 combined into a recombinant SOD) full sequence gene ( sod-combinant ) clones. The stability of the enzyme in aqueous solution and freeze-dried powder stored at room temperature was determined.

[0125] 1. Cloning construction, protein purification and enzyme activity determination of superoxide dismutase (SOD) gene

[0126] The specific method is shown in 2 of Example 1.

[0127] 2. Stability of superoxide dismutase in aqueous solution

[0128] Take 10ml of enzyme solution with a concentration of 50mg / ml and store it in an airtight container at room temperature (25°C). At different times (15 days, 30 days, 180 days, 360 days), the activity of superoxide dismutase was measured, and the protein precipitation of the enzyme was observed. The enzyme activity ...

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Abstract

The invention relates to a group of amino acid sequences capable of improving SOD (superoxide dismutase) stress resistance and stability in an aqueous solution and an application thereof. The amino acid sequences are a group of N-terminated amino acid sequences of 13 special thermophilic Fe / Mn-SOD originated from Geobacillus. The group of amino acid sequences has the characteristics that 1) 101-344 amino acids in sequence length before soda functional domains of Fe / Mn-SOD proteins are unknown in function; 2) the amino acid sequences has 85-100% homology thereamong; and 3) the amino acid sequences comprise one or two special shared repeated sequences Geo-N-repeat. The group of amino acid sequences has a decisive effect on the stress resistance and the stability in the aqueous solution of the Fe / Mn-SOD originated from Geobacillus, can be widely applied to transforming other SOD (particularly traditional industrial SOD) and the stress resistance and the stability in the aqueous solution are obviously improved.

Description

technical field [0001] The invention relates to a group of amino acid sequences capable of improving SOD stress resistance and stability in aqueous solution and applications thereof. Background technique [0002] Superoxide dismutase (superoxide dismutase, EC.1.5.1.1, SOD) is a kind of metalloenzyme, which widely exists in various organisms such as animals, plants and microorganisms. It was isolated from bovine red blood cells by Mann and Keilin in 1938. In 1969, Mecord and Fridovich discovered that the protein could catalyze the removal of superoxide anion radicals (O 2- ) reaction, is an important oxygen free radical scavenger, which can balance the oxygen free radicals in the body, so as to avoid adverse reactions caused by the excessive concentration of superoxide anion free radicals in the body. According to the combined metal ions, it can be divided into three types: Fe-SOD, Mn-SOD, and Cu / ZnSOD. SOD has the functions of anti-inflammation, anti-virus, anti-radiation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02
CPCC12N9/0089C12Y115/01001
Inventor 王威李洺畅
Owner 北京迈恩泰生物科技有限公司
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